27411, a novel human PGP synthase

ABSTRACT

The present invention relates to a newly identified human PGP synthase. The invention also relates to polynucleotides encoding the PGP synthase. The invention further relates to methods using the PGP synthase polypeptides and polynucleotides as a target for diagnosis and treatment in PGP synthase-mediated or -related disorders. The invention further relates to drug-screening methods using the PGP synthase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the PGP synthase polypeptides and polynucleotides. The invention further relates to procedures for producing the PGP synthase polypeptides and polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/185,517 filed Feb. 28, 2000, which is herebyincorporated in its entirety by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to newly identified humanphosphatidylglycerolphosphate (PGP) synthase belonging to the family ofmammalian PGP synthases.

[0003] The invention also relates to polynucleotides encoding the PGPsynthase. The invention further relates to methods using the PGPsynthase polypeptides and polynucleotides as a target for diagnosis andtreatment in PGP synthase-mediated or -related disorders. The inventionfurther relates to drug-screening methods using the PGP synthasepolypeptides and polynucleotides to identify agonists and antagonistsfor diagnosis and treatment. The invention further encompasses agonistsand antagonists based on the PGP synthase polypeptides andpolynucleotides. The invention further relates to procedures forproducing the PGP synthase polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0004] Cardiolipin is a dimeric phospholipid which plays an importantrole in mitochondrial biogenesis and function. It is required foractivity of several mitochondrial enzymes and possibly for the transportof proteins into the mitochondria in eukaryotes (Minskoff, S. et al.(1997) Biochimica et Biophysica Acta 1348: 187-191). Cardiolipin appearsto be involved either directly or indirectly, in the modulation of anumber of cellular processes including the activation of mitochondrialenzymes and the production of energy by oxidative phosphorylation(Hatch, G. (1998) International J. of Mol. Medicine 1: 33-41).

[0005] Cardiolipin is found in animals, plants, and fungi. In mammals itis found exclusively in mitochondria. Cardiolipin is the principalpolyglycerophospholipid found in the heart and most mammalian tissues(Hatch, G. (1998) International J. of Molec. Medicine 1:33-41). Thebiosynthetic pathway of cardiolipin has been well studied in yeasts. Thefirst enzyme in the cardiolipin biosynthetic pathway isphosphatidylglycerolphosphate synthase (PGP synthase). PGP synthase is akey enzyme in the pathway as it catalyzes the committed first step inthe pathway.

[0006] The biosynthesis of cardiolipin occurs in 3 enzymatic steps. Inthe first step, PGP synthase catalyzes the formation ofphosphatidylglycerolphosphate (PGP) from phosphatidyl-CMP(CDP-diacylglycerol, CDP-DG) and glycerol 3-phosphate. PGP is thendephosphorylated to phosphatidylglycerol (PG) by PGP phosphatase.Finally, in eukaryotes cardiolipin is synthesized from PG and anothermolecule of CDP-DG in a reaction catalyzed by cardiolipin synthase.

[0007] Cardiolipin appears to be essential for the function of severalenzymes of oxidative phosphorylation. (Hatch, G. (1996) Molecular andCellular Biochemistry 159:139-148). Also, cardiolipin has beenimplicated in the role of many enzymatic activities, including but notlimited to: (1) cytochrome c oxidase, (2) carnitine acylcarnitinetranslocase, (3) mitochondrial protein import, and (4) binding of matrixCa⁺² (Kawasaki,K. (1999) J. of Biological Chemistry, Vol. 274,No.3,1828-1834).

[0008] There must be stringent levels of control of the enzymes involvedin cardiolipin metabolism in the heart in order to maintain theappropriate content and molecular species composition of thephospholipid. The maintenance of cardiolipin content and molecularcomposition in cardiac mitochondria is essential for proper cardiacfunction (Hatch, G. (1998) International J. of Mol. Medicine 1:33-41).

[0009] Phosphatidylglycerol (PG) and cardiolipin (CL) are the mostwidely distributed glycerophosphatides in the membrane lipids ofanimals, plants and microbes (Hostletler, K. Y. (1982) in Phospholipids(Hawthorne and Ansell, eds) pp.215-261, Elsevier/North HollandBiomedical Press, Amsterdam).

[0010] PG is localized in many intracellular locations as a component ofphospholipids, representing less than 1% of total lipid phosphorous,except in the lung where it represents about 10% of the totalphospholipids (Mason, R. J. et al., (1980) Biochim. Biophys. Acta 617:36-50). PG serves as an important component of the pulmonary surfactantin the lung (Ohtsuka et al., (1993) J. Biol. Chem. Vol.268:22908-22913). CL is localized primarily in the mitochondria andappears to be essential for the function of several enzymes of oxidativephosphorylation. CL is essential for production of energy for the heartto beat (Hatch, G. M. (1996) Molecular and Cellular Biochemistry, 159:139-148).

[0011] PGP synthase has been extensively studied and characterized intwo evolutionarily divergent yeasts, Saccharomyces cerevisiae andSchizosaccharomyces pombe. PGP synthase has been purified to homogeneityfrom S. pombe (Minskoff, S. et al. (1997) Biochimica et Biophysica Acta1348: 187-191). In contrast to the second and third enzymes of thecardiolipin biosynthetic pathway, PGP synthase activity is highlyregulated both by cross-pathway control and by factors affectingmitochondrial development.

[0012] PGP synthase has been shown to be controlled by two sets offactors: cross-pathway control and factors affecting mitochondrialdevelopment. Cross-pathway control of phosphatidylinositol andphosphatidylcholine control is characterized by three parameters. First,the availability of the water-soluble phospholipid precursor inositolcontrols expression of phospholipid biosynthetic enzymes. Second,inositol repression of phospholipid biosynthesis occurs only if cellscan synthesize phosphatidyl-choline. Third, inositol repression ismediated by the INO2-INO4-OPI1 regulatory genes. PGP synthase isregulated by inositol. However, it is not subject to control by theINO2-INO4-OPI1 regulatory genes. PGP synthase activity is decreased 3-5fold in Saccharomyces cerevisiae cells grown in the presence of inositol(Greenberg, M. L. et al., (1988) Mol. Cell. Biol. 8: 4773-4779).

[0013] PGP synthase is commonly referred to as glycerophosphatephosphatidyl-transferase (E.C. 2.7.8.5). It catalyzes a substitutedphospho group transfer. The natural substrate of the enzyme isCDP-1,2-diacyl-sn-glycerol and glycerol 3-phosphate (involved in thesynthesis of phosphatidylgylcerol). Different cofactors and prostheticgroups which have been shown to be important for maximal PGP synthaseactivity include, but are not limited to: Triton X-100,phosphatidylethanolamine and phosphatidylinositol. Different metal/saltswhich have been shown to be important for PGP synthase activity include,but are not limited to: Mn⁺², Mg⁺², Ca⁺², Co⁺², and Ba⁺².

[0014] PGP synthases in two different yeasts (S. cerevisiae and S.pombe) were found to be sensitive to thioreactive compounds and have arequirement for divalent cations (Minskoff, S. et al. (1997) Biochimicaet Biophysica Acta 1348:187-191).

[0015] Inhibitors of PGP synthase have been shown to include, but arenot limited to: liponucleotide, CDPdiacylglycerol, glycerol 3-phosphate,thioreactive agents, calcium, inositol, Triton X-100, magnesium,cadmium, zinc, copper, and mercury(http://www.expasy.ch/cgi-bin/enzyme-search-ec). As one example, PGPsynthase activity was shown to decrease 3 to 5 fold in S. cerevisiaecells grown in the presence of inositol.

[0016] PGP synthase activity can be assayed by determining theconversion of [¹⁴C(U)] glycerol 3-phosphate to phosphatidyl[¹⁴C(U)]glycerol 3-phosphate as describedby Cao et al. (Cao et al.(1994) LIPIDS, Vol. 29, no.7, pp.475-480).

[0017] Chinese hamster ovary (CHO) cells defective in PGP synthaseproduction have been studied to better elucidate the role of the enzymein the biosynthesis of PG and CL (Ohtsuka, T. et al., (1993) J. Biol.Chem. Vol.268, No.30, pp. 22908-22913). Ohtsuka et al. developed arapidautoradiographic screening assay for detecting PGP synthase activity inthe lysates of Chinese hamster ovary cell colonies immobilized onpolyester, as described by Raetz et al. (Raetz et al., (1982) Proc.Natl. Acad. Sci. U.S.A. 79: 3223-3227). The Ohtsuka study confirmed therole of PGP synthase in the biosynthesis of PG and its essential role inthe growth of CHO cells. The results provided direct evidence for theformation of PG in vivo and that PG is a major metabolic precursor forthe biosynthesis of cellular CL.

[0018] Recent research has focused on the generation of a PGP-synthasedefective mutant in CHO-K1 cells (Kawasaki, K. et al. (1999) J. Biol.Chem. Vol. 274:1828-1834). Kawasaki et al. isolated a Chinese hamsterovary (CHO) cDNA encoding a putative protein similar in sequence to theyeast PGS1 gene product, PGP synthase. The CHO PGS1 cDNA encoded aprotein having high amino acid homology with the yeast PGS1.Transfection of CHO-K1 cells with CHO PGS1 cDNA in E.coli resulted in ahighly elevated PGP synthase activity level. Moreover, when the CHO PGS1was introduced into a mutant PGS-S (a temperature-sensitive mutantdefective in PGP synthase), the mutant recovered normal biosynthesis andcellular content of PG and CL. The results demonstrated the CHO PGS1cDNA encodes a PGP synthase. (Kawasaki, K. et al. (1999) J. Biol. Chem.Vol. 274, No.3, pp.1828-1834). The cloned CHO PGS1 cDNA was able tocomplement the mitochondrial defect as well as the biosynthetic defectsin CL and PG biosynthesis.

[0019] Moreover, there is an apparent difference in the molecularmechanisms of the PGP synthases between eukaryotic and prokaryoticorganisms. The eukaryotic PGP synthases most likely utilize a ping-pongreaction mechanism, in contrast to the prokaryotic PGP synthases thatemploy abi-bi reaction mechanism (Dryden, S. (1996) J. Bacteriol. 178:1030-1038). PGP synthase is an essential enzyme in bacteria (Heacock, P.N. et al., (1987) J. Biol. Chem. 262:13044-13049). Presumably, thisdifference in reaction mechanism between eukaryotic and prokaryotic PGPsynthases might represent a target for antibacterial agents (Kawasaki,K. et al. (1999) J. Biol. Chem. Vol. 274, No.3, pp. 1828-1⁸34).

[0020] PGP synthases are important as relates to cardiolipin metabolismin aging and thyroid dysfunction. Aging and hypothyroidism are twoconditions associated with mitochondrial dysfunction and cardiolipindeficiency. (Schlame, M. et al., (1997) Biochimica et Biophysica Acta,1348:207-213). In both cases, mitochondrial cardiolipin deficiency couldbe correlated with a decrease in metabolite transport activity acrossmitochondrial membrane. As relates to the aging process, it has beensuggested that cardiolipin deficiency is the cause of reduced metabolitetransport due to changes in the membrane environment of the carrierproteins (Paradies et al. (1992) Biochim. Biophys. Acta 1103: 324-326).

[0021] Conversely, hyperthyroidism is characterized by mitochondria withincreased cardiolipin content and increased metabolite transportactivities (Paradies (1990) Biochim. Biophys. Acta 1019:133-136).Thyroxine is a well-known stimulator of mitochondrial biogenesis; it isknown to increase the number of mitochondria as well as enhance theirperformance.

[0022] Accordingly, PGP synthases are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize novel PGP synthases and tissuesand disorders in which PGP synthases are differentially expressed. Thepresent invention advances the state of the art by providing a novelhuman PGP synthase and tissues and disorders in which expression of thehuman PGP synthase is relevant. Accordingly, the invention providesmethods directed to expression of the PGP synthase.

SUMMARY OF THE INVENTION

[0023] It is an object of the invention to identify a novel PGPsynthase.

[0024] It is a further object of the invention to provide novel PGPsynthase polypeptides that are useful as reagents or targets in assaysapplicable to treatment and diagnosis of PGP synthase-mediated or-related disorders.

[0025] It is a further object of the invention to providepolynucleotides corresponding to the novel PGP synthase polypeptidesthat are useful as targets and reagents in PGP synthase assaysapplicable to treatment and diagnosis of PGP synthase-mediated or-related disorders and useful for producing novel PGP synthasepolypeptides by recombinant methods.

[0026] A specific object of the invention is to identify compounds thatact as agonists and antagonists and modulate the expression of the novelPGP synthase.

[0027] A further specific object of the invention is to providecompounds that modulate expression of the PGP synthase for treatment anddiagnosis of PGP synthase-related disorders.

[0028] The invention is thus based on the identification of a novelhuman PGP synthase. The amino acid sequence for PGP synthase is shown inSEQ ID NO:2. The nucleotide sequence for PGP synthase is shown in SEQ IDNO:1.

[0029] The invention provides isolated PGP synthase polypeptides,including a polypeptide having the amino acid sequence shown in SEQ IDNO:2, or the amino acid sequences encoded by the cDNAs deposited asPatent Deposit Nos. PTA-2011 and PTA-2340.

[0030] The invention also provides isolated PGP synthase nucleic acidmolecules having the sequence shown in SEQ ID NO:1, SEQ ID NO:3, or inthe deposited cDNAs.

[0031] The invention also provides variant polypeptides having an aminoacid sequence that is substantially homologous to the amino acidsequence shown in SEQ ID NO:2 or encoded by the deposited cDNAs.

[0032] The invention also provides variant nucleic acid sequences thatare substantially homologous to the nucleotide sequence shown in SEQ IDNO:1 or in the deposited cDNAs.

[0033] The invention also provides fragments of the polypeptides shownin SEQ ID NO:2 and nucleotide sequence shown in SEQ ID NO:1 as well assubstantially homologous fragments of the polypeptides or nucleic acids.

[0034] The invention further provides nucleic acid constructs comprisingthe nucleic acid molecules described herein. In a preferred embodiment,the nucleic acid molecules of the invention are operatively linked to aregulatory sequence.

[0035] The invention also provides vectors and host cells for expressingthe PGP synthase nucleic acid molecules and polypeptides, andparticularly recombinant vectors and host cells.

[0036] The invention also provides methods of making the vectors andhost cells and methods for using them to produce the PGP synthasenucleic acid molecules and polypeptides.

[0037] The invention also provides antibodies or antigen-bindingfragments thereof that selectively bind the PGP synthase polypeptidesand fragments.

[0038] The invention also provides methods of screening for compoundsthat modulate expression or activity of the PGP synthase polypeptides ornucleic acid (RNA or DNA).

[0039] The invention also provides a process for modulating PGP synthasepolypeptide or nucleic acid expression or activity, especially using thescreened compounds. Modulation may be used to treat conditions relatedto aberrant activity or expression of the PGP synthase polypeptides ornucleic acids.

[0040] The invention also provides assays for determining the activityof or the presence or absence of the PGP synthase polypeptides ornucleic acid molecules in a biological sample, including for diseasediagnosis.

[0041] The invention also provides assays for determining the presenceof a mutation in the polypeptides or nucleic acid molecules, includingfor disease diagnosis.

[0042] In still a further embodiment, the invention provides a computerreadable means containing the nucleotide and/or amino acid sequences ofthe nucleic acids and polypeptides of the invention, respectively.

DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and the deducedamino acid sequence (SEQ ID NO:2) of the novel PGP synthase. The PGPsynthase coding sequence, nucleotides 315-1985 of SEQ ID NO: 1,constitutes SEQ ID NO:3.

[0044]FIG. 2 shows an analysis of the PGP synthase amino acid sequence:αβturn and coil regions; hydrophilicity; amphipathic regions; flexibleregions; antigenic index; and surface probability plot.

[0045]FIG. 3 shows a hydrophobicity plot of the PGP synthase amino acidsequence (SEQ ID NO:2). Relative hydrophobic residues are shown abovethe dashed horizontal line, and relative hydrophilic residues are belowthe dashed horizontal line. The cysteine residues (cys) and Nglycosylation site (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The numbers corresponding to the amino acidsequence (shown in SEQ ID NO:2) of human 27411 are indicated.Polypeptides of the invention include fragments which include: all or apart of a hydrophobic sequence (a sequence above the dashed line); orall or part of a hydrophilic fragment (a sequence below the dashedline). Other fragments include a cysteine residue or an N-glycosylationsite. A signal peptide is predicted from about amino acid 1 to aboutamino acid 68. Also shown are the predicted transmembrane segments ofthe full length protein from about amino acid 51 to about amino acid 73and from about amino acid 469 to about amino acid 485. A predictedtransmembrane segment for the presumed mature peptide is from aboutamino acid 402 to about amino acid 418.

[0046]FIG. 4 shows an analysis of the PGP synthase open reading framefor amino acids (SEQ ID NO:2) corresponding to specific functionalsites. N-glycosylation sites are found from about amino acid 213 toabout amino acid 216, from about amino acid 236 to about amino acid 239,and from about amino acid 390 to about amino acid 393. Cyclic AMP andcGMP-dependent protein kinase phosphorylation sites are found from aboutamino acid 46 to about amino acid 49 and from about amino acid 172 toabout 175. Protein kinase C phosphorylation sites are found from aboutamino acid 35 to about amino acid 37, from about amino acid 243 to aboutamino acid 245, and from about amino acid 313 to about amino acid 315.Casein kinase II phosphorylation sites are found from about amino acid102 to about amino acid 105, from about amino 143 to about amino acid146, from about amino acid 333 to about amino acid 336, from about aminoacid 374 to about amino acid 377, and from about amino acid 402 to aboutamino acid 405. A tyrosine kinase phosphorylation site is found fromabout amino acid 344 to about amino acid 352. N-myristoylation sites arefound from about amino acid 19 to about amino acid 24, from about aminoacid 91 to about amino acid 96, from about amino acid 234 to about aminoacid 239, from about amino acid 423 to about amino acid 428, and fromabout amino acid 527 to about amino acid 532. An amidation site is foundfrom about amino acid 170 to about amino acid 173.

[0047]FIG. 5 depicts relative expression of 27411 in various humanorgans and cell types: artery (column 1), diseased artery (column 2),vein (column 3), coronary smooth muscle cells (column 4), HUVEC(umbilical vein endothelial cells) (column 5), hemangioma (column 6),heart (column 7), congestive heart failure heart (column 8), kidney(column 9), skeletal muscle (column 10), adipose (column 11), pancreas(column 12), primary osteoblasts (column 13), differentiated osteoclasts(column 14), skin (column 15), spinal cord (column 16), brain cortex(column 17), brain hypothalamus (column 18), nerve (column 19), dorsalroot ganglion (column 20), breast (column 21), breast tumor (column 22),ovary (column 23), ovarian tumor (column 24), prostate (column 25),prostate tumor (column 26), salivary glands (column 27), colon (column28), colon tumor (column 29), lung (column 30), lung tumor (column 31),chronic obstructive pulmonary disease lung (column 32), spleen (column33), tonsil (column 34), lymph node (column 35), small intestine (column36), macrophages (column 37), synovium (column 38), mononuclear bonemarrow cells (column 39), activated peripheral blood mononuclear cells(column 40), neutrophils (column 41), megakaryocytes (column 42), anderythroid tissue (column 43). Tissues or cell types were normal unlessindicated otherwise. Expression levels were determined by quantitativeRT-PCR (Taqman® brand quantitative PCR kit, Applied Biosystems). Thesequantitative RT-PCR reactions were performed according to the kitmanufacturer's instructions.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated by reference for the purpose of describing and disclosingcell lines, vectors, and methodologies which are reported in thepublications which might be used in connection with the invention.Nothing is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

[0049] “Nucleic acid sequence” as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments andportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single-or double-stranded, and represents the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring, recombinant orsynthetic molecules.

[0050] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, aminoacid sequence and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein.

[0051] PGP synthase as used herein, refers to the amino acid sequencesof substantially purified PGP synthase obtained from any species,particularly mammalian, including bovine, ovine, porcine, murine,equine, and preferably human, from any source whether natural,synthetic, semi-synthetic, or recombinant.

[0052] A “deletion” as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acids ornucleotide residues, are absent.

[0053] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues.

[0054] A “substitution” as used herein, refers to the replacement of oneor more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0055] The term “biologically active” as used herein, refers to aprotein having structural, regulatory, or biochemical functions of thePGP synthase. Also “immunologically” active refers to the capability ofthe natural, recombinant, or synthetic PGP synthase, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

[0056] The term “agonist” as used herein, refers to a molecule which,when bound to the synthase causes a change in PGP synthase whichmodulates activity of PGP synthase. Agonists may include proteins,nucleic acids, carbohydrates or any other molecules.

[0057] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which blocks or modulates the biological activity of PGPsynthase. Antagonists may include proteins, nucleic acids,carbohydrates, or any other molecules.

[0058] The term “modulate” as used herein, refers to a change in thebiological level or activity of PGP synthase. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics of PGP synthase to its substrate or effector molecule,or any other change in the biological, functional, or immunologicalproperties of PGP synthase.

[0059] The term “derivative” as used herein, refers to the chemicalmodifications of a nucleic acid encoding PGP synthase or the encoded PGPsynthase. Illustrations of such modifications would be replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativewould encode a polypeptide which retains essential biologicalcharacteristics of the natural molecule.

[0060] Polypeptides

[0061] The invention is based on the identification of a novel PGPsynthase and the polynucleotide sequence encoding the PGP synthase.

[0062] The invention thus relates to a novel PGP synthase having theamino acid sequence shown in FIG. 1 or the amino acid sequence shown inSEQ ID NO:2, or the amino acid sequences encoded by the deposited cDNAsas Patent Deposit Nos. PTA-201 I or PTA-2340.

[0063] Plasmids containing the nucleotide sequences of the inventionwere deposited with the Patent Depository of the American Type CultureCollection (ATCC), Manassas, Va., on Jun. 9, 2000 and Aug. 10, 2000 andassigned Patent Deposit Nos. PTA-2011 and PTA-2340, respectively. Thedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms. Thedeposit is provided as a convenience to those of skill in the art and isnot an admission that a deposit is required under 35 U.S.C. § 112. Thedeposited sequences, as well as the polypeptides encoded by thesequence, are incorporated herein by reference and controls in the eventof any conflict, such as a sequencing error, with description in thisapplication.

[0064] “PGP synthase polypeptide” or “PGP synthase protein” refers tothe polypeptide in SEQ ID NO:2, or the polypeptide encoded by thedeposited cDNA. The term “PGP synthase protein” or “PGP synthasepolypeptide”, however, further includes the numerous variants describedherein, as well as fragments derived from the full-length PGP synthaseand variants. By “variants” is intended proteins or polypeptides havingan amino acid sequence that is at least about 60%, 65%, or 70%,preferably about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO:2. Variantsalso include polypeptides encoded by the cDNA inserts of the plasmidsdeposited with the ATCC as Patent Deposit Number PTA-20 11 or PTA-2340,or polypeptides encoded by a nucleic acid molecule that hybridizes tothe nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, or a complementthereof, under stringent conditions. In another embodiment, a variant ofan isolated polypeptide of the present invention differs, by at least 1,but less than 5, 10, 20, 50, or 100 amino acid residues from thesequence shown in SEQ ID NO:2. If alignment is needed for thiscomparison the sequences should be aligned for maximum identity.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences. Such variants retain the functional activity ofthe PGP synthase like proteins of the invention. Variants includepolypeptides that differ in amino acid sequence due to natural allelicvariation or mutagenesis.

[0065] PGP synthases are found in most mammalian tissues with thehighest concentrations in the heart, lung, and liver(http://www.expasy.ch/enzyme).

[0066] The present invention thus provides isolated or purifiedpolypeptides of the PGP synthase and variants and fragments thereof.

[0067] Based on a Blast search, highest homology to the PGP synthase ofthe invention was shown to phosphatidylglycerophosphate synthase fromCricetulus griseus (Genbank Acc. No. AB016930). The polypeptide of theinvention is 93% identical to the C. griseusphosphatidylglycerophosphate synthase in the region from amino acids 4to 556 of SEQ ID NO:2. The nucleotide sequence of the invention is 87%identical to the C. griseus phosphatidylglycerophosphate synthasenucleotide sequence in the region from nucleotides 326-1991 of SEQ IDNO:1.

[0068] As used herein, a polypeptide is said to be “isolated” or“purified” when it is substantially free of cellular material when it isisolated from recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can bejoinedto another polypeptide with which itis not normally associated in a cell and still be considered “isolated”or “purified.”

[0069] The PGP synthase polypeptides can be purified from mammaliantissues (McMurray, W. C. et al., (1978) Can J. Biochem. 56, 414-419). Itis understood, however, that preparations in which the polypeptide isnot purified to homogeneity are useful and considered to contain anisolated form of the polypeptide. The critical feature is that thepreparation allows for the desired function of the polypeptide, even inthe presence of considerable amounts of other components. Thus, theinvention encompasses various degrees of purity.

[0070] In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the PGP synthase having less thanabout 30% (by dry weight) other proteins (i.e., contaminating protein),less than about 20% other proteins, less than about 10% other proteins,or less than about 5% other proteins. When the polypeptide isrecombinantly produced, it can also be substantially free of culturemedium, i.e., culture medium represents less than about 20%, less thanabout 10%, or less than about 5% of the volume of the proteinpreparation.

[0071] A PGP synthase polypeptide is also considered to be isolated whenit is part of a membrane preparation or is purified and thenreconstituted with membrane vesicles or liposomes.

[0072] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the PGP synthase polypeptide inwhich it is separated from chemical precursors or other chemicals thatare involved in its synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of the polypeptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

[0073] In one embodiment, the PGP synthase polypeptides comprise theamino acid sequences shown in SEQ ID NO:2. However, the invention alsoencompasses sequence variants. Variants include a substantiallyhomologous protein encoded by the same genetic locus in an organism,i.e., an allelic variant.

[0074] Variants also encompass proteins derived from other genetic lociin an organism, but having substantial homology to the PGP synthase ofSEQ ID NO:2. Variants also include proteins substantially homologous tothe PGP synthase but derived from another organism, i.e., an ortholog.Variants also include proteins that are substantially homologous to thePGP synthase that are produced by chemical synthesis. Variants alsoinclude proteins that are substantially homologous to the PGP synthasethat are produced by recombinant methods. Variants retain the functionalactivity of the PGP synthase like polypeptides set forth in SEQ ID NO:2.It is understood, however, that variants exclude any amino acidsequences disclosed prior to the invention.

[0075] As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity. A substantially homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence hybridizing to the nucleic acid sequence, orportion thereof, of the sequence shown in SEQ ID NO:1 or SEQ ID NO:3under stringent conditions as more fully described below. To determinethe percent identity of two amino acid sequences, or of two nucleic acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0076] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) is using a Blossum 62 scoringmatrix with a gap open penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

[0077] The percent identity between two amino acid or nucleotidesequences can be determined using the algorithm of E. Meyers and W.Miller (1989) CABIOS 4:11-17 which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

[0078] The nucleic acid and protein sequences described herein can beused as a “query sequence” to perform a search against public databasesto, for example, identify other family members or related sequences.Such searches can be performed using the NBLAST and XBLAST programs(version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the 27411nucleic acid molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to the 27411 protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.n1m.nih.gov.

[0079] The invention also encompasses polypeptides having a lower degreeof identity but having sufficient similarity so as to perform one ormore of the same functions performed by the PGP synthase. Similarity isdetermined by conserved amino acid substitution. Such substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Conservative substitutions arelikely to be phenotypically silent. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr, exchange of the acidic residues Asp and Glu, substitutionbetween the amide residues Asn and Gln, exchange of the basic residuesLys and Arg and replacements among the aromatic residues Phe, Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990). TABLE 1 Conservative Amino Acid Substitutions. AromaticPhenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine ValinePolar Glutamine Asparagine Basic Arginine Lysine Histidine AcidicAspartic Acid Glutamic Acid Small Alanine Serine Threonine MethionineGlycine

[0080] A variant polypeptide can differ in amino acid sequence by one ormore substitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these. Variant polypeptides canbe fully functional or can lack function in one or more activities.Variants include those having alterations that affect interaction withany of the substrates or effector molecules, including but not limitedto those disclosed herein or that affect the function of the PGPsynthase that normally results from such interaction. For example,variants of the PGP synthase can have an altered binding affinity forthe substrates, CDP-diacylglycerol and glycerol 3-phosphate.

[0081] Another useful variation provides a fusion protein in which oneor more domains or subregions are operationally fused to one or moredomains or subregions from another PGP synthase. Specifically, a domainor subregion can be introduced that alters the substrate specificitiesor the rate of the enzymatic reaction.

[0082] Fully functional variants typically contain only conservativevariations or variations in non-critical residues or in non-criticalregions. Functional variants can also contain substitution of similaramino acids, which results in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyaffect function to some degree.

[0083] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0084] As indicated, variants can be naturally occurring or can be madeby recombinant means or chemical synthesis to provide useful and novelcharacteristics for the PGP synthase polypeptide. This includespreventing immunogenicity from pharmaceutical formulations by preventingprotein aggregation.

[0085] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al. (1985) Science244:1081-1085). The latter procedure introduces single alanine mutationsat every residue in the molecule. The resulting mutant molecules arethen tested for PGP synthase activity, such as the binding affinity forthe substrates or determining the catalytic constants for substitutedphospho group transfer between CDP-diacylglycerol and glycerol3-phosphate. Sites that are critical for substrate binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al. (1992) J.Mol. Biol. 224:899-904; de Vos et al. (1992) Science 255:306-312).

[0086] The assays for PGP synthase enzyme activity are well known in theart and can be found for example, in Ohtsuka et al.(1993) J. Biol. Chem.Vol. 268, No.30, 22908-22913). Substantial homology can be to the entirenucleic acid or amino acid sequence or to fragments of these sequences.

[0087] The invention thus also includes polypeptide fragments of the PGPsynthase. Fragments can be derived from the amino acid sequences shownin SEQ ID NO:2. However, the invention also encompasses fragments of thevariants of the PGP synthase as described herein.

[0088] The fragments to which the invention pertains, however, are notto be construed as encompassing fragments that may be disclosed prior tothe present invention. Accordingly, a fragment of the PGP synthase cancomprise at least about 20, 25, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, or 556 contiguous amino acids. Fragments canretain one or more of the biological activities of the protein, forexample the ability to bind the substrate or the ability to catalyze thesubstituted phospho group transfer. Alternatively, fragments can be usedas an immunogen to generate PGP synthase antibodies.

[0089] Biologically active fragments (peptides which are, for example,5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400,450, 500,550, or 556 amino acids in length) can comprise a domain or motifincluding a substrate binding site, catalytic binding site and sites forglycosylation, protein kinase C phosphorylation, Casein kinase IIphosphorylation, cyclic AMP and cGMP-dependent phosphorylation, tyrosinekinase phosphorylation and N-myristoylation. Further possible fragmentsmay include sites important for cellular and subcellular targeting.

[0090] Such domains or motifs can be identified by means of routinecomputerized homology searching procedures.

[0091] Fragments, for example, can extend in one or both directions fromthe functional site to encompass 5, 10, 15, 20,30, 40, 50, or up to 100amino acids. Further, fragments can include sub-fragments of thespecific domains mentioned above, which sub-fragments retain thefunction of the domain from which they are derived.

[0092] These regions can be identified by well-known methods involvingcomputerized homology analysis.

[0093] The invention also provides fragments with immunogenicproperties. These contain an epitope-bearing portion of the PGP synthaseor PGP synthase variants. These epitope-bearing peptides are useful toraise antibodies that bind specifically to an PGP synthase polypeptideor region or fragment. These peptides can contain at least 5, 10, atleast 15, or between at least about 15 to about 30 amino acids.

[0094] Non-limiting examples of antigenic polypeptides that can be usedto generate antibodies include but are not limited to peptides derivedfrom an extracellular site. Regions having a high antigenicity index areshown in FIG. 2 for the PGP synthase. However, intracellularly-madeantibodies (“intrabodies”) are also encompassed, which would recognizeintracellular peptide regions.

[0095] The epitope-bearing PGP synthase polypeptides may be produced byany conventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci.USA 82:5131-5135). Simultaneous multiple peptide synthesis is describedin U.S. Pat. No. 4,631,211.

[0096] Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the PGP synthase fragment and an additional region fused tothe carboxyl terminus of the fragment.

[0097] The invention thus provides chimeric or fusion proteins. Thesecomprise a PGP synthase peptide sequence operatively linked to aheterologous peptide having an amino acid sequence not substantiallyhomologous to the PGP synthase. “Operatively linked” indicates that thePGP synthase peptide and the heterologous peptide are fused in-frame.The heterologous peptide can be fused to the N-terminus or C-terminus ofthe PGP synthase or can be internally located. In the case where anexpression cassette contains two protein-coding regions joined in acontiguous manner in the same reading frame, the encoded polypeptide isherein defined as a “heterologous polypeptide” or a “chimericpolypeptide” or a “fusion polypeptide”. As used herein, a PGP synthase“heterologous protein” or “chimeric protein” or “fusion protein”comprises a PGP synthase polypeptide operatively linked to a non-PGPsynthase polypeptide.

[0098] In one embodiment the fusion protein does not affect PGP synthasefunction per se. For example, the fusion protein can be a GST-fusionprotein in which the PGP synthase sequences are fused to the C-terminusof the GST sequences. Other types of fusion proteins include, but arenot limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL4 fusions, poly-Hisfusions and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinant PGP synthase. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence. Therefore, in another embodiment, the fusion protein containsa heterologous signal sequence at its N-terminus.

[0099] EP-A-O 464 533 discloses fusion proteins comprising variousportions of immunoglobulin constant regions. The Fc is useful in therapyand diagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists (Bennett et al.(1995) J. Mol. Recog. 8:52-58 (1995) and Johanson et al. J. Biol. Chem.270:9459-9471). Thus, this invention also encompasses soluble fusionproteins containing an PGP synthase polypeptide and various portions ofthe constant regions of heavy or light chains of immunoglobulins ofvarious subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin isthe constant part of the heavy chain of human IgG, particularly IgGI,where fusion takes place at the hinge region. For some uses it isdesirable to remove the Fc after the fusion protein has been used forits intended purpose, for example when the fusion protein is to be usedas antigen for immunizations. In a particular embodiment, the Fc partcan be removed in a simple way by a cleavage sequence, which is alsoincorporated and can be cleaved with factor Xa.

[0100] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al. (1992) Current Protocols in Molecular Biology). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). An PGP synthase-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the PGP synthase.

[0101] Another form of fusion protein is one that directly affects PGPsynthase functions. Accordingly, a PGP synthase polypeptide isencompassed by the present invention in which one or more of the PGPsynthase domains (or parts thereof) has been replaced by homologousdomains (or parts thereof) from another PGP synthase. Accordingly,various permutations are possible. For example, the binding or catalyticdomain, or subregion thereof, can be replaced with the domain orsubregion from another PGP synthase or another phosphatidyl transferase.Thus, chimeric PGP synthases can be formed in which one or more of thenative domains or subregions has been replaced by another.

[0102] Additionally, chimeric PGP synthase proteins can be produced inwhich one or more functional sites is derived from a different PGPsynthase or isoform. It is understood however that sites could bederived from other PGP synthases that occur in the mammalian genome butwhich have not yet been discovered or characterized. Such sites includebut are not limited to the catalytic site and substrate binding sites,and other functional sites disclosed herein.

[0103] It is further recognized that the nucleic acid sequences of theinvention can be altered to contain codons, which are preferred, ornon-preferred, for a particular expression system. For example, thenucleic acid can be one in which at least one altered codon, andpreferably at least 10%, or 20% of the codons have been altered suchthat the sequence is optimized for expression in E. coli, yeast, human,insect, or CHO cells. Methods for determining such codon usage are wellknown in the art.

[0104] The isolated PGP synthase can be purified from cells thatnaturally express it, including but not limited to heart, lung and liveras well as the tissues shown in FIG. 5. The PGP synthase of the presentinvention can also be purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods.

[0105] In one embodiment, the protein is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the PGPsynthase polypeptide is cloned into an expression vector, the expressionvector introduced into a host cell and the protein expressed in the hostcell. The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally-occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in polypeptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art.

[0106] Accordingly, the polypeptides also encompass derivatives oranalogs in which a substituted amino acid residue is not one encoded bythe genetic code, in which a substituent group is included, in which themature polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or in which the additional amino acids are fused to the maturepolypeptide, such as a leader or secretory sequence or a sequence forpurification of the mature polypeptide or a pro-protein sequence.

[0107] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0108] Such modifications are well-known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2nded., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth.Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N. Y. Acad. Sci.663:48-62).

[0109] As is also well known, polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of post-translation events, including naturalprocessing events and events brought about by human manipulation whichdo not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translational natural processesand by synthetic methods.

[0110] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. Blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally-occurring andsynthetic polypeptides. For instance, the aminoterminal residue ofpolypeptides made in E. coli, prior to proteolytic processing, almostinvariably will be N-formylmethionine.

[0111] The modifications can be a function of how the protein is made.For recombinant polypeptides, for example, the modifications will bedetermined by the host cell posttranslational modification capacity andthe modification signals in the polypeptide amino acid sequence.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often cany out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications.

[0112] The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a givenpolypeptide may contain more than one type of modification.

[0113] Polypeptide Uses

[0114] The PGP synthase polypeptides are useful for producing antibodiesspecific for the PGP synthase, regions, or fragments. Regions having ahigh antigenicity index score are shown in FIG. 2.

[0115] The PGP synthase polypeptides are useful for biological assaysrelated to PGP synthase. Such assays involve any of the known PGPsynthase functions or activities or properties useful for diagnosis andtreatment of PGP synthase-related conditions, including CL and PGbiosynthesis.

[0116] The PGP synthase polypeptides are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the PGP synthase, as a biopsyor expanded in cell culture. In one embodiment, however, cell-basedassays involve recombinant host cells expressing the PGP synthase, suchas those disclosed in the background above.

[0117] Determining the ability of the test compound to interact with thePGP synthase can also comprise determining the ability of the testcompound to preferentially bind to the polypeptide as compared to theability of a known binding molecule (e.g. CDP-diacylglycerol andglycerol 3-phosphate) to bind to the polypeptide.

[0118] The polypeptides can be used to identify compounds that modulatePGP synthase activity. Such compounds, for example, can increase ordecrease the affinity or rate of binding to the substratesCDP-diacylglycerol and glycerol 3-phosphate, compete with the substratesfor binding to the PGP synthase, or displace substrates bound to the PGPsynthase. Such compounds can also increase or decrease the enzymaticactivity of the PGP synthase. Compounds that modulate PGP synthaseactivity include, but are not limited to, liponucleotides, CDPdiacylglycerol, glycerol 3-phosphate (Hirabayashi et al. (1976)Biochemistry 15: 5205-5211), thioreactive agents (Carman et al. (1984)J. Food Biochem 8:321-333), inositol (Bleasdale et al. (1982) Biochim.Biophys. Acta 710:377-390), and Ca²⁺(Dowhan et al. (1992) MethodsEnzymol 71:313-321).

[0119] The PGP synthase of the present invention and appropriatevariants and fragments can be used in high-throughput screens to assaycandidate compounds for the ability to bind to the PGP synthase. Thesecompounds can be further screened against a functional PGP synthase todetermine the effect of the compound on the PGP synthase activity.Compounds can be identified that activate (agonist) or inactivate(antagonist) the PGP synthase to a desired degree. Modulatory methodscan be performed in vitro (e.g., by culturing the cell with the agent)or, alternatively, in vivo (e.g., by administering the agent to asubject).

[0120] The PGP synthase polypeptides can be used to screen a compoundfor the ability to stimulate or inhibit interaction between the PGPsynthase protein and a target molecule that normally interacts with thePGP synthase protein. The target can be a cofactor, metal ion, or PGAsynthase substrate. Different cofactors and prosthetic groups which havebeen shown to be important for maximal PGP synthase activity include,but are not limited to Triton X-100, phosphatidylethanolamine andphosphatidylinositol.

[0121] Different metal/salts which have been shown to be important forPGP synthase activity include, but are not limited to Mn⁺², Mg⁺², Ca⁺²,Co⁺², and Ba⁺². The assay includes the steps of combining the PGPsynthase protein with a candidate compound under conditions that allowthe PGP synthase protein or fragment to interact with the targetmolecule, and to detect the formation of a complex between the PGPsynthase protein and the target or to detect the biochemical consequenceof the interaction with the PGP synthase and the target.

[0122] Determining the ability of the PGP synthase to bind to a targetmolecule can also be accomplished using a technology such as real-timeBimolecular Interaction Analysis (BIA). Sjolander et al. (1991) Anal.Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore™). Changes in the optical phenomenon surfaceplasmon resonance (SPR) can be used as an indication of real-timereactions between biological molecules.

[0123] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, K. S. (1997)Anticancer Drug Des. 12:145).

[0124] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries ofcompounds may be presented in solution (e.g., Houghten (1992)Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 97:6378-6382), (Felici(1991) J. Mol. Biol. 222:301-310); (Ladnersupra).

[0125] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84;Houghten et al. (1991) Nature 354:84-86) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0126] One candidate compound is a soluble full-length PGP synthase orfragment that competes for substrate binding, including but not limitedto those disclosed herein. Other candidate compounds include mutant PGPsynthases or appropriate fragments containing mutations that affect PGPsynthase function and thus compete for substrates, e.g.,CDP-diacylglcerol and glycerol 3-phosphate. Accordingly, a fragment thatcompetes for substrate binding, for example with a higher affinity, or afragment that binds substrate(s) but does not catalyze the phospho grouptransfer is encompassed by the invention.

[0127] The invention provides other end points to identify compoundsthat modulate (stimulate or inhibit) PGP synthase activity. The assaystypically involve an assay of events that result from a substitutedphospho group transfer that indicate PGP synthase activity. Thus, theexpression of genes that are up- or down-regulated in response to thePGP synthase enzyme can be assayed. In one embodiment, the regulatoryregion of such genes can be operably linked to a marker that is easilydetectable, such as luciferase. Additionally, measurements of metabolitetransport across mitochondrial membranes and mitochondrial cardiolipincontent can serve as parameters to quantify PGP synthase activity.

[0128] Any of the biological or biochemical functions mediated by thePGP synthase can be used as an endpoint assay. These include all of thebiochemical or biological events described herein, in the referencescited herein and incorporated by reference for these events, and otherPGP synthase functions known to those of ordinary skill in the art.

[0129] Binding and/or activating compounds can also be screened by usingchimeric PGP synthase proteins in which one or more domains, sites, andthe like, as disclosed herein, or parts thereof, can be replaced bytheir heterologous counterparts derived from other PGP synthases. Forexample, a substrate binding region or cofactor binding region can beused that interacts with a different substrate or cofactor specificityand/or affinity than the native PGP synthase. Alternatively, aheterologous targeting sequence can replace the native targetingsequence. This will result in different subcellular or cellularlocalization. As a further alternative, sites that are responsible fordevelopmental, temporal, or tissue specificity can be replaced byheterologous sites such that the PGP synthase can be detected underconditions of specific developmental, temporal, or tissue-specificexpression.

[0130] The PGP synthase polypeptides are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the PGP synthase. Thus, a compound is exposed to a PGP synthasepolypeptide under conditions that allow the compound to bind or tootherwise interact with the polypeptide. Soluble PGP synthasepolypeptide is also added to the mixture. If the test compound interactswith the soluble PGP synthase polypeptide, it decreases the amount ofcomplex formed or activity from the PGP synthase target. This type ofassay is particularly useful in cases in which compounds are sought thatinteract with specific regions of the PGP synthase. Thus, the solublepolypeptide that competes with the target PGP synthase region isdesigned to contain peptide sequences corresponding to the region ofinterest.

[0131] Another type of competition-binding assay can be used to discovercompounds that interact with specific functional sites and inhibit PGPsynthase. As an example, the substrates (CDP-diacylglycerol and glycerol3-phosphate) and a candidate compound can be added to a sample of PGPsynthase. Compounds that interact with PGP synthase at the same site asthe substrates will reduce the amount of complex formed between the PGPsynthase and the substrates. One example of a group of compounds thataffect PGP synthase activity are thioreactive agents. Additionalinhibitors of PGP synthase include: liponucleotide, inositol, TritonX-100, and the divalent cations of magnesium, calcium, cadmium, zinc,mercury, and copper at certain critical millimolar concentrations.(http://www.expasy.ch/enzyme).

[0132] To perform cell free drug screening assays, it is desirable toimmobilize either the PGP synthase, or fragment, or its target moleculeto facilitate separation of complexes from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.

[0133] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/ PGP synthase fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the cell lysates (e.g., ³⁵S-labeled) andthe candidate compound, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly, or in the supernatant after the complexes is dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of PGP synthase-binding proteinfound in the bead fraction quantitated from the gel using standardelectrophoretic techniques. For example, either the polypeptide or itstarget molecule can be immobilized utilizing conjugation of biotin andstreptavidin using techniques well known in the art. Alternatively,antibodies reactive with the protein but which do not interfere withbinding of the protein to its target molecule can be derivatized to thewells of the plate, and the protein trapped in the wells by antibodyconjugation. Preparations of a PGP synthase-binding target component anda candidate compound are incubated in the PGP synthase-presenting wellsand the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the PGP synthase targetmolecule, or which are reactive with PGP synthase and compete with thetarget molecule; as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

[0134] Modulators of PGP synthase activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by PGP synthase, by treating cells that express the PGPsynthase. These methods of treatment include the steps of administeringthe modulators of PGP synthase activity in a pharmaceutical compositionas described herein, to a subject in need of such treatment. Treatmentis defined as the application or administration of a therapeutic agentto a patient, or application or administration of a therapeutic agent toan isolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. “Subject”, as used herein, can refer to amammal, e.g. a human, or to an experimental or animal or disease model.The subject can also be a non-human animal, e.g. a horse, cow, goat, orother domestic animal. A therapeutic agent includes, but is not limitedto, small molecules, peptides, antibodies, ribozymes and antisenseoligonucleotides.

[0135] The PGP synthases are expressed in tissues including, but notlimited to heart, lung, liver, and the tissues shown in FIG. 5.

[0136] Hence the PGP synthase of the present invention is relevant totreating disorders involving these tissues.

[0137] Disorders involving the lung include, but are not limited to,congenital anomalies-atelectasis; diseases of vascular origin, such aspulmonary congestion and edema, including hemodynamic pulmonary edemaand edema caused by microvascular injury, adult respiratory distresssyndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

[0138] Disorders involving the liver include, but are not limited to,hepatic injury; jaundice and cholestasis, such as bilirubin and bileformation; hepatic failure and cirrhosis, such as cirrhosis, portalhypertension, including ascites, portosystemic shunts, and splenomegaly;infectious disorders, such as viral hepatitis, including hepatitis A-Einfection and infection by other hepatitis viruses, clinicopathologicsyndromes, such as the carrier state, asymptomatic infection, acuteviral hepatitis, chronic viral hepatitis, and fulminant hepatitis;autoimmune hepatitis; drug- and toxin-induced liver disease, such asalcoholic liver disease; inborn errors of metabolism and pediatric liverdisease, such as hemochromatosis, Wilson disease, a₁-antitrypsindeficiency, and neonatal hepatitis; intrahepatic biliary tract disease,such as secondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

[0139] Disorders involving the heart, include but are not limited to,heart failure, including but not limited to, cardiac hypertrophy,left-sided heart failure, and right-sided heart failure; ischemic heartdisease, including but not limited to angina pectoris, myocardialinfarction, chronic ischemic heart disease, and sudden cardiac death;hypertensive heart disease, including but not limited to, systemic(left-sided) hypertensive heart disease and pulmonary (right-sided)hypertensive heart disease; valvular heart disease, including but notlimited to, valvular degeneration caused by calcification, such ascalcific aortic stenosis, calcification of a congenitally bicuspidaortic valve, and mitral annular calcification, and myxomatousdegeneration of the mitral valve (mitral valve prolapse), rheumaticfever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

[0140] Disorders involving blood vessels include, but are not limitedto, responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

[0141] Disorders involving red cells include, but are not limited to,anemias, such as hemolytic anemias, including hereditary spherocytosis,hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B12 deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

[0142] Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

[0143] Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones), and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

[0144] Disorders involving the pancreas include those of the exocrinepancreas such as congenital anomalies, including but not limited to,ectopic pancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas, and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

[0145] Bone-forming cells include the osteoprogenitor cells,osteoblasts, and osteocytes. The disorders of the bone are complexbecause they may have an impact on the skeleton during any of its stagesof development. Hence, the disorders may have variable manifestationsand may involve one, multiple or all bones of the body. Such disordersinclude, congenital malformations, achondroplasia and thanatophoricdwarfism, diseases associated with abnormal matrix such as type 1collagen disease, osteoporosis, Paget disease, rickets, osteomalacia,high-tumover osteodystrophy, low-turnover of aplastic disease,osteonecrosis, pyogenic osteomyelitis, tuberculous osteomyelitism,osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma,chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma,fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignantfibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal tumor,giant cell tumor, and metastatic tumors.

[0146] Disorders involving the brain include, but are not limited to,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-bome (Arbo) viral encephalitis,Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degeneration,multiple system atrophy, including striatonigral degeneration,Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntingtondisease; spinocerebellar degenerations, including spinocerebellarataxias, including Friedreich ataxia, and ataxia-telangectasia,degenerative diseases affecting motor neurons, including amyotrophiclateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

[0147] Disorders of the breast include, but are not limited to,disorders of development; inflammations, including but not limited to,acute mastitis, periductal mastitis, periductal mastitis (recurrentsubareolar abscess, squamous metaplasia of lactiferous ducts), mammaryduct ectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

[0148] Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

[0149] Disorders involving the ovary include, for example, polycysticovarian disease, Stein-Leventhal syndrome, Pseudomyxoma peritonei andstromal hyperthecosis; ovarian tumors such as, tumors of coelomicepithelium, serous tumors, mucinous tumors, endometeriod tumors, clearcell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

[0150] Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

[0151] Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0152] Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

[0153] Disorders involving the small intestine include the malabsorptionsyndromes such as, celiac sprue, tropical sprue (postinfectious sprue),whipple disease, disaccharidase (lactase) deficiency,abetalipoproteinemia, and tumors of the small intestine includingadenomas and adenocarcinoma.

[0154] In normal bone marrow, the myelocytic series (polymorphoneuclearcells) make up approximately 60% of the cellular elements, and theerythrocytic series, 20-30%. Lymphocytes, monocytes, reticular cells,plasma cells and megakaryocytes together constitute 10-20%. Lymphocytesmake up 5-15% of normal adult marrow. In the bone marrow, cell types areadd mixed so that precursors of red blood cells (erythroblasts),macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclearleucocytes (myeloblasts), and lymphocytes (lymphoblasts) can be visiblein one microscopic field. In addition, stem cells exist for thedifferent cell lineages, as well as a precursor stem cell for thecommitted progenitor cells of the different lineages. The various typesof cells and stages of each would be known to the person of ordinaryskill in the art and are found, for example, on page 42 (FIGS. 2-8) ofImmunology, Imunopathology and Immunity, Fifth Edition, Sell et al.Simon and Schuster (1996), incorporated by reference for its teaching ofcell types found in the bone marrow. According, the invention isdirected to disorders arising from these cells. These disorders includebut are not limited to the following: diseases involving hematopoieticstem cells, committed lymphoid progenitor cells; lymphoid cellsincluding B and T-cells; committed myeloid progenitors, includingmonocytes, granulocytes, and megakaryocytes; and committed erythroidprogenitors. These include but are not limited to the leukemias,including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiatedleukemias; erythroleukemia, megakaryoblastic leukemia, monocytic;[leukemias are encompassed with and without differentiation]; chronicand acute lymphoblastic leukemia, chronic and acute lymphocyticleukemia, chronic and acute myelogenous leukemia, lymphoma, myelodysplastic syndrome, chronic and acute myeloid leukemia, myelomonocyticleukemia; chronic and acute myeloblastic leukemia, chronic and acutemyelogenous leukemia, chronic and acute promyelocytic leukemia, chronicand acute myelocytic leukemia, hematologic malignancies ofmonocyte-macrophage lineage, such as juvenile chronic myelogenousleukemia; secondary AML, antecedent hematological disorder; refractoryanemia; aplastic anemia; reactive cutaneous angioendotheliomatosis;fibrosing disorders involving altered expression in dendritic cells,disorders including systemic sclerosis, E-M syndrome, epidemic toxic oilsyndrome, eosinophilic fasciitis localized forms of scleroderma, keloid,and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma;carcinoma, including primary head and neck squamous cell carcinoma;sarcoma, including kaposi's sarcoma; fibroadanoma and phyllodes tumors,including mammary fibroadenoma; stromal tumors; phyllodes tumors,including histiocytoma; erythroblastosis; neurofibromatosis; diseases ofthe vascular endothelium; demyelinating, particularly in old lesions;gliosis, vasogenic edema, vascular disease, Alzheimer's and Parkinson'sdisease; T-cell lymphomas; B-cell lymphomas.

[0155] Diseases of the skin, include but are not limited to, disordersof pigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

[0156] Disorders involving the tonsils include, but are not limited to,tonsillitis, Peritonsillar abscess, squamous cell carcinoma, dyspnea,hyperplasia, follicular hyperplasia, reactive lymphoid hyperplasia,non-Hodgkin's lymphoma and B-cell lymphoma.

[0157] Examples of cellular proliferative and/or differentiativedisorders include cancer, e.g., carcinoma, sarcoma, metastatic disordersor hematopoietic neoplastic disorders, e.g., leukemias. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast, and liver origin.

[0158] As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

[0159] The terms “cancer” or “neoplasms” include malignancies of thevarious organ systems, such as affecting lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

[0160] The term “carcinoma” is art recognized and refers to malignanciesof epithelial or endocrine tissues including respiratory systemcarcinomas, gastrointestinal system carcinomas, genitourinary systemcarcinomas, testicular carcinomas, breast carcinomas, prostaticcarcinomas, endocrine system carcinomas, and melanomas. Exemplarycarcinomas include those forming from tissue of the cervix, lung,prostate, breast, head and neck, colon and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures.

[0161] The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation.

[0162] The 27411 nucleic acid and protein of the invention can be usedto treat and/or diagnose a variety of proliferative disorders. E.g.,such disorders include hematopoietic neoplastic disorders. As usedherein, the term “hematopoietic neoplastic disorders” includes diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Preferably, the diseases arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L.(1991) Crit. Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignanciesinclude, but are not limited to acute lymphoblastic leukemia (ALL) whichincludes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemiallymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

[0163] PGP synthases are important as relates to cardiolipin metabolismin the aging process and thyroid dysfunction. Aging and hypothyroidismare two conditions associated with mitochondrial dysfunction andcardiolipin deficiency. (Schlane, M. et al., (1997) Biochimica etBiophysica Acta, 1348:207-213). Also, hyperthyroidism is characterizedby mitochondria with increased cardiolipin content and increasedmetabolite transport activities. (Paradies, G. et al., (1992) Biochim.Biophys. Acta, 1019:133-136). Therefore, PGP synthases may prove to beuseful clinical tools for treating any of these processes andconditions.

[0164] The PGP synthase polypeptides are thus useful for treating a PGPsynthase-associated disorder characterized by aberrant expression oractivity of an PGP synthase. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed or cited herein), or combination of agents that modulates(e.g., upregulates or downregulates) expression or activity of theprotein. In another embodiment, the method involves administering thePGP synthase as therapy to compensate for reduced or aberrant expressionor activity of the protein.

[0165] Methods for treatment include but are not limited to the use ofsoluble PGP synthase or fragments of the PGP synthase protein thatcompete for substrate binding, or interfere with the reaction mediatedby the PGP synthase polypeptide. These PGP synthase or fragments canhave a higher affinity for the target so as to provide effectivecompetition.

[0166] Stimulation of activity is desirable in situations in which theprotein is abnormally downregulated and/or in which increased activityis likely to have a beneficial effect. Likewise, inhibition of activityis desirable in situations in which the protein is abnormallyupregulated and/or in which decreased activity is likely to have abeneficial effect. In one example of such a situation, a subject has adisorder characterized by aberrant development or cellulardifferentiation. In another example, the subject has a proliferativedisease (e.g., cancer).

[0167] In yet another aspect of the invention, the proteins of theinvention can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identifyother proteins (captured proteins) which bind to or interact with theproteins of the invention and modulate their activity.

[0168] The PGP synthase polypeptides also are useful to provide a targetfor diagnosing a disease or predisposition to disease mediated by thePGP synthase, including, but not limited to, diseases involving tissuesin which the PGP synthase is expressed, as described herein.Accordingly, methods are provided for detecting the presence, or levelsof, the PGP synthase in a cell, tissue, or organism. The method involvescontacting a biological sample with a compound capable of interactingwith the PGP synthase such that the interaction can be detected.

[0169] One agent for detecting PGP synthase is an antibody capable ofselectively binding to PGP synthase. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0170] The PGP synthase also provides a target for diagnosing activedisease, or predisposition to disease, in a patient having a variant PGPsynthase. Thus, PGP synthase can be isolated from a biological sampleand assayed for the presence of a genetic mutation that results in anaberrant protein. This includes amino acid substitution, deletion,insertion, rearrangement, (as the result of aberrant splicing events),and inappropriate post-translational modification. Analytic methodsinclude altered electrophoretic mobility, altered tryptic peptidedigest, altered PGP synthase activity in cell-based or cell-free assay,alteration in substrate binding, altered substituted phospho grouptransfer, altered antibody-binding pattern, altered isoelectric point,direct amino acid sequencing, and any other of the known assaytechniques useful for detecting mutations in a protein in general or inan PGP synthase specifically.

[0171] In vitro techniques for detection of PGP synthase include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, the proteincan be detected in vivo in a subject by introducing into the subject alabeled anti-PGP synthase antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. Particularlyuseful are methods, which detect the allelic variant of the PGP synthaseexpressed in a subject, and methods, which detect fragments of the PGPsynthase in a sample.

[0172] The PGP synthase polypeptides are also useful in pharmacogenomicanalysis. Pharmacogenomics deal with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, e.g., Eichelbaum, M. (1996)Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985, and Linder, M. W.(1997) Clin. Chem. 43(2):254-266. The clinical outcomes of thesevariations result in severe toxicity of therapeutic drugs in certainindividuals or therapeutic failure of drugs in certain individuals as aresult of individual variation in metabolism. Thus, the genotype of theindividual can determine the way a therapeutic compound acts on the bodyor the way the body metabolizes the compound. Further, the activity ofdrug metabolizing enzymes affects both the intensity and duration ofdrug action. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype.

[0173] The discovery of genetic polymorphisms in some drug metabolizingenzymes has explained why some patients do not obtain the expected drugeffects, show an exaggerated drug effect, or experience serious toxicityfrom standard drug dosages. Polymorphisms can be expressed in thephenotype of the extensive metabolizer and the phenotype of the poormetabolizer. Accordingly, genetic polymorphism may lead to allelicprotein variants of the PGP synthase in which one or more of the PGPsynthase functions in one population is different from those in anotherpopulation. The polypeptides thus allow a target to ascertain a geneticpredisposition that can affect treatment modality. Thus, in an PGPsynthase-based treatment, polymorphism may give rise to catalyticregions that are more or less active. Accordingly, dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing the polymorphism. As an alternative togenotyping, specific polymorphic polypeptides could be identified.

[0174] The PGP synthase polypeptides are also useful for monitoringtherapeutic effects during clinical trials and other treatment. Thus,the therapeutic effectiveness of an agent that is designed to increaseor decrease gene expression, protein levels or PGP synthase activity canbe monitored over the course of treatment using the PGP synthasepolypeptides as an end-point target. The monitoring can be, for example,as follows: (i) obtaining a pre-administration sample from a subjectprior to administration of the agent; (ii) detecting the level ofexpression or activity of the protein in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of theprotein in the post-administration samples; (v) comparing the level ofexpression or activity of the protein in the pre-administration samplewith the protein in the post-administration sample or samples; and (vi)increasing or decreasing the administration of the agent to the subjectaccordingly.

[0175] Antibodies

[0176] The invention also provides antibodies that selectively bind tothe PGP synthase and its variants and fragments. An antibody isconsidered to selectively bind, even if it also binds to other proteinsthat are not substantially homologous with the PGP synthase. These otherproteins share homology with a fragment or domain of the PGP synthase.This conservation in specific regions gives rise to antibodies that bindto both proteins by virtue of the homologous sequence. In this case, itwould be understood that antibody binding to the PGP synthase is stillselective.

[0177] To generate antibodies, an isolated PGP synthase polypeptide isused as an immunogen to generate antibodies using standard techniquesfor polyclonal and monoclonal antibody preparation. Either thefull-length protein or antigenic peptide fragment can be used. Regionshaving a high antigenicity index are shown in FIG. 2.

[0178] Antibodies are preferably prepared from these regions or fromdiscrete fragments in these regions. However, antibodies can be preparedfrom any region of the peptide as described herein. A preferred fragmentproduces an antibody that diminishes or completely prevents substrate orcofactor binding or prevents the transfer of the phospho group.Antibodies can be developed against the entire PGP synthase or domainsof the PGP synthase as described herein. Antibodies can also bedeveloped against specific functional sites as disclosed herein.

[0179] The antigenic peptide can comprise a contiguous sequence of atleast 12, 14, 15, or 30 amino acid residues. In one embodiment,fragments correspond to regions that are located on the surface of theprotein, e.g., hydrophilic regions. These fragments are not to beconstrued, however, as encompassing any fragments, which may bedisclosed prior to the invention.

[0180] Antibodies can be polyclonal or monoclonal. An intact antibody,or a fragment thereof (e.g. Fab or F(ab′)₂) can be used.

[0181] Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0182] An appropriate immunogenic preparation can be derived fromnative, recombinantly expressed, or chemically synthesized peptides.

[0183] Antibody Uses

[0184] The antibodies can be used to isolate a PGP synthase by standardtechniques, such as affinity chromatography or immunoprecipitation. Theantibodies can facilitate the purification of the natural PGP synthasefrom cells and recombinantly produced PGP synthase expressed in hostcells.

[0185] The antibodies are useful to detect the presence of PGP synthasein cells or tissues to determine the pattern of expression of the PGPsynthase among various tissues in an organism and over the course ofnormal development.

[0186] The antibodies can be used to detect PGP synthase in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression.

[0187] The antibodies can be used to assess abnormal tissue distributionor abnormal expression during development.

[0188] Antibody detection of circulating fragments of the full lengthPGP synthase can be used to identify PGP synthase turnover.

[0189] Further, the antibodies can be used to assess PGP synthaseexpression in disease states such as in active stages of the disease orin an individual with a predisposition toward disease related to PGPsynthase function. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, or level of expression of thePGP synthase protein, the antibody can be prepared against the normalPGP synthase protein. If a disorder is characterized by a specificmutation in the PGP synthase, antibodies specific for this mutantprotein can be used to assay for the presence of the specific mutant PGPsynthase. However, intracellularly-made antibodies (“intrabodies”) arealso encompassed, which would recognize intracellular PGP synthasepeptide regions.

[0190] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Antibodies can be developed against the whole PGP synthase or portionsof the PGP synthase.

[0191] The diagnostic uses can be applied, not only in genetic testing,but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting PGP synthase expressionlevel or the presence of aberrant PGP synthase and aberrant tissuedistribution or developmental expression, antibodies directed againstthe PGP synthase or relevant fragments can be used to monitortherapeutic efficacy.

[0192] Antibodies accordingly can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.

[0193] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic PGP synthase can be usedto identify individuals that require modified treatment modalities.

[0194] The antibodies are also useful as diagnostic tools as animmunological marker for aberrant PGP synthase analyzed byelectrophoretic mobility, isoelectric point, tryptic peptide digest, andother physical assays known to those in the art.

[0195] The antibodies are also useful for tissue typing. Thus, where aspecific PGP synthase has been correlated with expression in a specifictissue, antibodies that are specific for this PGP synthase can be usedto identify a tissue type.

[0196] The antibodies are also useful in forensic identification.Accordingly, where an individual has been correlated with a specificgenetic polymorphism resulting in a specific polymorphic protein, anantibody specific for the polymorphic protein can be used as an aid inidentification.

[0197] The antibodies are also useful for inhibiting PGP synthasefunction, for example, blocking substrate binding or disrupting transferof the phospho group between CDP-diacylglycerol and glycerol3-phosphate.

[0198] These uses can also be applied in a therapeutic context in whichtreatment involves inhibiting PGP synthase function. An antibody can beused, for example, to block substrate binding. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact PGP synthase associated with a cell.

[0199] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. For an overview of thistechnology for producing human antibodies, see Lonberg et al. (1995)Int. Rev. Immunol. 13:65-93. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, e.g., U.S. Pat.No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.

[0200] The invention also encompasses kits for using antibodies todetect the presence of an PGP synthase protein in a biological sample.The kit can comprise antibodies such as a labeled or labelable antibodyand a compound or agent for detecting PGP synthase in a biologicalsample; means for determining the amount of PGP synthase in the sample;and means for comparing the amount of PGP synthase in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectPGP synthase.

[0201] Polynucleotides

[0202] The nucleotide sequence in SEQ ID NO:1 was obtained by sequencingthe deposited human cDNA. Accordingly, the sequence of the depositedclones are controlling as to any discrepancies between the two and anyreference to the sequences of SEQ ID NO:1, includes reference to thesequences of the deposited cDNA.

[0203] The specifically disclosed cDNAs comprise the coding region and5′ and 3′ untranslated sequences in SEQ ID NO:1.

[0204] The invention provides isolated polynucleotides encoding thenovel PGP synthase. The term “PGP synthase polynucleotide” or “PGPsynthase nucleic acid” refers to the sequences shown in SEQ ID NO:1, SEQID NO:3, or in the deposited cDNAs. The term “PGP synthasepolynucleotide” or “PGP synthase nucleic acid” further includes variantsand fragments of the PGP synthase polynucleotides. Generally, nucleicacid molecules that are fragments of the 27411 nucleic acid comprise atleast 15, 20, 38, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2686 nucleotides or up to the number ofnucleotides present in a full-length human PGP synthase-like nucleotidesequence disclosed herein (for example, 2686 nucleotides for SEQ IDNO:1) depending upon the intended use. Alternatively, a nucleic acidmolecule that is a fragment of a 27411-like nucleotide sequence of thepresent invention comprises a nucleotide sequence consisting ofnucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000,2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600,2600-2686 of SEQ ID NO:1.

[0205] An “isolated” PGP synthase nucleic acid is one that is separatedfrom other nucleic acid present in the natural source of the PGPsynthase nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the PGP synthase nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.However, there can be some flanking nucleotide sequences, for example upto about 5 kb. The important point is that the PGP synthase nucleic acidis isolated from flanking sequences such that it can be subjected to thespecific manipulations described herein, such as recombinant expression,preparation of probes and primers, and other uses specific to the PGPsynthase nucleic acid sequences.

[0206] Moreover, an “isolated” nucleic acid molecule, such as a cDNA orRNA molecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0207] In some instances, the isolated material will form part of acomposition (for example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0208] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0209] In some instances, the isolated material will form part of acomposition (or example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0210] The PGP synthase polynucleotides can encode the mature proteinplus additional amino or carboxyterminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0211] The PGP synthase polynucleotides include, but are not limited to,the sequence encoding the mature polypeptide alone, the sequenceencoding the mature polypeptide and additional coding sequences, such asa leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature polypeptide, with or withoutthe additional coding sequences, plus additional non-coding sequences,for example introns and non-coding 5′ and 3′ sequences such astranscribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thepolynucleotide may be fused to a marker sequence encoding, for example,a peptide that facilitates purification.

[0212] PGP synthase polynucleotides can be in the form of RNA, such asmRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0213] PGP synthase nucleic acid can comprise the nucleotide sequencesshown in SEQ ID NO:1 and SEQ ID NO:3 corresponding to human PGP synthasecDNA.

[0214] In one embodiment, the PGP synthase nucleic acid comprises onlythe coding region.

[0215] The invention further provides variant PGP synthasepolynucleotides, and fragments thereof, that differ from the nucleotidesequences shown in SEQ ID NO:1 due to degeneracy of the genetic code andthus encode the same protein as that encoded by the nucleotide sequencesshown in SEQ ID NO:1 or SEQ ID NO:3.

[0216] The invention also provides PGP synthase nucleic acid moleculesencoding the variant polypeptides described herein. Such polynucleotidesmay be naturally occurring, such as allelic variants (same locus),homologs (different locus), and orthologs (different organism), or maybe constructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to polynucleotides, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions.

[0217] Generally, nucleotide sequences variants of the invention willhave at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence disclosedherein. Variation can occur in either or both the coding and non-codingregions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0218] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a PGP synthase that is at least about 60-65%, 65-70%,typically at least about 70-75%, more typically at least about 80-85%,and most typically at least about 90-95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or a fragment ofthese sequences. Such nucleic acid molecules can readily be identifiedas being able to hybridize under stringent conditions, to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:3, or a fragment of thesesequences. It is understood that stringent hybridization does notindicate substantial homology where it is due to general homology, suchas poly A sequences, or sequences common to all or most proteins, allPGP synthase, all phospho group transferases. Moreover, it is understoodthat variants do not include any of the nucleic acid sequences that mayhave been disclosed prior to the invention.

[0219] As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred, example of stringenthybridization conditions are hybridization in 6×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6×sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by one or more washes in 0.2SSC, 0.1% SDS at 60° C. Preferably,stringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferred stringencyconditions (and the conditions that should be used if the practitioneris uncertain about what conditions should be applied to determine if amolecule is within a hybridization limitation of the invention) are 0.5MSodium Phosphate, 7% SDS at 65° C., followed by one or more washes at0.2×SSC, 1% SDS at 65° C. Preferably, an isolated nucleic acid moleculeof the invention that hybridizes under stringent conditions to thesequence of SEQ ID NO:1, or SEQ ID NO:3, corresponds to a naturallyoccurring nucleic acid molecule.

[0220] As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein).

[0221] As understood by those of ordinary skill, the exact conditionscan be determined empirically and depend on ionic strength, temperatureand the concentration of destabilizing agents such as forrnamide ordenaturing agents such as SDS. Other factors considered in determiningthe desired hybridization conditions include the length of the nucleicacid sequences, base composition, percent mismatch between thehybridizing sequences and the frequency of occurrence of subsets of thesequences within other non-identical sequences. Thus, equivalentconditions can be determined by varying one or more of these parameterswhile maintaining a similar degree of identity or similarity between thetwo nucleic acid molecules.

[0222] The present invention also provides isolated nucleic acids thatcontain a single or double stranded fragment or portion that hybridizesunder stringent conditions to the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:3 or the complement thereof. In one embodiment, the nucleicacid consists of a portion of the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:3 or the complement thereof.

[0223] It is understood that isolated fragments include any contiguoussequence not disclosed prior to the invention as well as sequences thatare substantially the same and which are not disclosed. Accordingly, ifa fragment is disclosed prior to the present invention, that fragment isnot intended to be encompassed by the invention. When a sequence is notdisclosed prior to the present invention, an isolated nucleic acidfragment is at least about 12, preferably at least about 15, 18, 20, 23or 25 nucleotides, and can be 30, 40, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600 or more nucleotides inlength. Longer fragments, for example, 30 or more nucleotides in length,which encode antigenic proteins or polypeptides described herein areuseful.

[0224] For PGP synthase, for example, nucleotide sequences from about 1to about 285, from about 1992 to about 2041 and from about 2562 to about2643 are especially relevant and encompass fragments of 5-10, 10-15,15-20, 20-25, etc., as disclosed herein. The nucleotide sequence fromabout 1 to about 1991 encompasses fragments greater than about 315, 325,345, 355 or 365 nucleotides; the nucleotide sequence from about 1074 toabout 2689 encompasses fragments greater than 167, 175, 185, 195, or 205nucleotides; and the nucleotide sequence from about 2507 to about 2689encompasses fragments greater than 28, 35, 40, 45, 50, or 55nucleotides.

[0225] The fragment can be single or double-stranded and can compriseDNA or RNA. The fragment can be derived from either the coding or thenon-coding sequence.

[0226] In another embodiment an isolated PGP synthase nucleic acidencodes the entire coding region. In another embodiment the isolated PGPsynthase nucleic acid encodes a sequence corresponding to the matureprotein. For example, the mature form of the PGP synthase is from aboutamino acid 68 to the last amino acid. Other fragments include nucleotidesequences encoding the amino acid fragments described herein.

[0227] Thus, PGP synthase nucleic acid fragments further includesequences corresponding to the domains described herein, subregions alsodescribed, and specific functional sites. PGP synthase nucleic acidfragments also include combinations of the domains, segments, and otherfunctional sites described above. A person of ordinary skill in the artwould be aware of the many permutations that are possible.

[0228] Where the location of the domains or sites have been predicted bycomputer analysis, one of ordinary skill would appreciate that the aminoacid residues constituting these domains can vary depending on thecriteria used to define the domains.

[0229] However, it is understood that a PGP synthase fragment includesany nucleic acid sequence that does not include the entire gene.

[0230] The invention also provides PGP synthase nucleic acid fragmentsthat encode epitope bearing regions of the PGP synthase proteinsdescribed herein.

[0231] Nucleic acid fragments, according to the present invention, arenot to be construed as encompassing those fragments that may have beendisclosed prior to the invention.

[0232] Polynucleotide Uses

[0233] The nucleic acid fragments of the invention provide probes orprimers in assays such as those described below. “Probes” areoligonucleotides that hybridize in a base-specific manner to acomplementary strand of nucleic acid. Such probes include polypeptidenucleic acids, as described in Nielsen et al. (1991) Science254:1497-1500. Typically, a probe comprises a region of nucleotidesequence that hybridizes under highly stringent conditions to at leastabout 15, typically about 20-25, and more typically about 40, 50 or 75consecutive nucleotides of the nucleic acid sequence shown in SEQ IDNO:1 and the complements thereof. More typically, the probe furthercomprises a label, e.g., radioisotope, fluorescent compound, enzyme, orenzyme co-factor.

[0234] As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis using well-known methods (e.g., PCR, LCR) including, butnot limited to those described herein. The appropriate length of theprimer depends on the particular use, but typically ranges from about 15to 30 nucleotides. The term “primer site” refers to the area of thetarget DNA to which a primer hybridizes. The term “primer pair” refersto a set of primers including a 5′ (upstream) primer that hybridizeswith the 5′ end of the nucleic acid sequence to be amplified and a 3′(downstream) primer that hybridizes with the complement of the sequenceto be amplified.

[0235] The PGP synthase polynucleotides are thus useful for probes,primers, and in biological assays.

[0236] Where the polynucleotides are used to assess PGP synthaseproperties or functions, such as in the assays described herein, all orless than all of the entire cDNA can be useful. Assays specificallydirected to PGP synthase functions, such as assessing agonist orantagonist activity, encompass the use of known fragments. Further,diagnostic methods for assessing PGP synthase function can also bepracticed with any fragment, including those fragments that may havebeen known prior to the invention. Similarly, in methods involvingtreatment of PGP synthase dysfunction, all fragments are encompassedincluding those, which may have been known in the art.

[0237] The PGP synthase polynucleotides are useful as a hybridizationprobe for cDNA and genomic DNA to isolate a full-length cDNA and genomicclones encoding the polypeptides described in SEQ ID NO:2 and to isolatecDNA and genomic clones that correspond to variants producing the samepolypeptides shown in SEQ ID NO:2 or the other variants describedherein. Variants can be isolated from the same tissue and organism fromwhich the polypeptides shown in SEQ ID NO:2 were isolated, differenttissues from the same organism, or from different organisms. This methodis useful for isolating genes and cDNA that aredevelopmentally-controlled and therefore may be expressed in the sametissue or different tissues at different points in the development of anorganism.

[0238] The probe can correspond to any sequence along the entire lengthof the gene encoding the PGP synthase. Accordingly, it could be derivedfrom 5′ noncoding regions, the coding region, and 3′ noncoding regions.

[0239] The nucleic acid probe can be, for example, the full-length cDNAof SEQ ID NO:1 or a fragment thereof, such as an oligonucleotide of atleast 10-15, 15-20, 20-25, 25-30, 100, 250, or 500 nucleotides in lengthand sufficient to specifically hybridize under stringent conditions tomRNA or DNA.

[0240] Fragments of the polynucleotides described herein are also usefulto synthesize larger fragments or full-length polynucleotides describedherein. For example, a fragment can be hybridized to any portion of anmRNA and a larger or full-length cDNA can be produced.

[0241] The fragments are also useful to synthesize antisense moleculesof desired length and sequence.

[0242] Antisense nucleic acids of the invention can be designed usingthe nucleotide sequences of SEQ ID NO:1 or SEQ ID NO:3 and constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

[0243] Additionally, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670. PNAs can be further modified, e.g., to enhance theirstability, specificity or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. The synthesis of PNA-DNA chimeras can be performed as described inHyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res.24(17):3357-63, Mag et al. (1989) Nucleic Acids Res. 17:5973, andPeterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

[0244] The nucleic acid molecules and fragments of the invention canalso include other appended groups such as peptides (e.g., for targetinghost cell PGP synthase in vivo), or agents facilitating transport acrossthe cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/09810) or the blood brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).

[0245] The PGP synthase polynucleotides are also useful as primers forPCR to amplify any given region of a PGP synthase polynucleotide.

[0246] The PGP synthase polynucleotides are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the PGP synthase polypeptides. Vectorsalso include insertion vectors, used to integrate into anotherpolynucleotide sequence, such as into the cellular genome, to alter insitu expression of PGP synthase genes and gene products. For example, anendogenous PGP synthase coding sequence can be replaced via homologousrecombination with all or part of the coding region containing one ormore specifically introduced mutations.

[0247] The PGP synthase polynucleotides are also useful for expressingantigenic portions of the PGP synthase proteins.

[0248] The PGP synthase polynucleotides are also useful as probes fordetermining the chromosomal positions of the PGP synthasepolynucleotides by means of in situ hybridization methods, such as FISH.(For a review of this technique, see Verma et al. (1988) HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York),and PCR mapping of somatic cell hybrids. The mapping of the sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

[0249] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0250] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland et al.((1987) Nature 325:783-787).

[0251] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with a specified gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations, that are visible from chromosome spreads,or detectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0252] The PGP synthase polynucleotide probes are also useful todetermine patterns of the presence of the gene encoding the PGP synthaseand their variants with respect to tissue distribution, for example,whether gene duplication has occurred and whether the duplication occursin all or only a subset of tissues. The genes can be naturally occurringor can have been introduced into a cell, tissue, or organismexogenously.

[0253] The PGP synthase polynucleotides are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced fromgenes encoding the polynucleotides described herein.

[0254] The PGP synthase polynucleotides are also useful for constructinghost cells expressing a part, or all, of the PGP synthasepolynucleotides and polypeptides.

[0255] The PGP synthase polynucleotides are also useful for constructingtransgenic animals expressing all, or a part, of the PGP synthasepolynucleotides and polypeptides.

[0256] The PGP synthase polynucleotides are also useful for makingvectors that express part, or all, of the PGP synthase polypeptides.

[0257] The PGP synthase polynucleotides are also useful as hybridizationprobes for determining the level of PGP synthase nucleic acidexpression. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, PGP synthase nucleic acid in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the polypeptidesdescribed herein can be used to assess gene copy number in a given cell,tissue, or organism. This is particularly relevant in cases in whichthere has been an amplification of the PGP synthase genes.

[0258] Alternatively, the probe can be used in an in situ hybridizationcontext to assess the position of extra copies of the PGP synthasegenes, as on extrachromosomal elements or as integrated into chromosomesin which the PGP synthase gene is not normally found, for example as ahomogeneously staining region.

[0259] These uses are relevant for diagnosis of disorders involving anincrease or decrease in PGP synthase expression relative to normal, suchas a proliferative disorder or a differentiative or developmentaldisorder.

[0260] Disorders in which PGP synthase expression is relevant include,but are not limited to disease conditions associated with defectivecardiolipin (CL) and phosphatidylglycerol (PG) biosynthesis andmetabolism.

[0261] Tissues and/or cells in which 27411 is expressed are describedabove herein. As such, the gene is particularly relevant for thetreatment of disorders involving these tissues.

[0262] Furthermore, disorders in which 27411 expression is relevant aredisclosed herein above.

[0263] Thus, the present invention provides a method for identifying adisease or disorder associated with aberrant expression or activity ofPGP synthase nucleic acid, in which a test sample is obtained from asubject and nucleic acid (e.g., mRNA, genomic DNA) is detected, whereinthe presence of the nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantexpression or activity of the nucleic acid.

[0264] “Misexpression or aberrant expression”, as used herein, refers toa non-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

[0265] One aspect of the invention relates to diagnostic assays fordetermining nucleic acid expression as well as activity in the contextof a biological sample (e.g., blood, serum, cells, tissue) to determinewhether an individual has a disease or disorder, or is at risk ofdeveloping a disease or disorder, associated with aberrant nucleic acidexpression or activity. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated withexpression or activity of the nucleic acid molecules.

[0266] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0267] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express the PGP synthase, such as bymeasuring the level of a PGP synthase-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if thePGP synthase gene has been mutated.

[0268] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate PGP synthase nucleic acid expression(e.g., antisense, polypeptides, peptidomimetics, small molecules orother drugs). A cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of the mRNA inthe presence of the candidate compound is compared to the level ofexpression of the MRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Themodulator can bind to the nucleic acid or indirectly modulateexpression, such as by interacting with other cellular components thataffect nucleic acid expression.

[0269] Modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the gent to a subject) in patients or in transgenicanimals.

[0270] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the PGP synthase gene. The method typically includesassaying the ability of the compound to modulate the expression of thePGP synthase nucleic acid and thus identifying a compound that can beused to treat a disorder characterized by undesired PGP synthase nucleicacid expression.

[0271] The assays can be performed in cell-based and cell-free systems.Cell-based assays include cells naturally expressing the PGP synthasenucleic acid or recombinant cells genetically engineered to expressspecific nucleic acid sequences, for example those cited above and inthe background.

[0272] Alternatively, candidate compounds can be assayed in vivo inpatients or in transgenic animals.

[0273] The assay for PGP synthase nucleic acid expression can involvedirect assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the PGP synthase catalyzed reaction.Further, the expression of genes that are up- or down-regulated inresponse to the PGP synthase signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

[0274] Thus, modulators of PGP synthase gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof PGP synthase mRNA in the presence of the candidate compound iscompared to the level of expression of PGP synthase mRNA in the absenceof the candidate compound. The candidate compound can then be identifiedas a modulator of nucleic acid expression based on this comparison andbe used, for example to treat a disorder characterized by aberrantnucleic acid expression. When expression of mRNA is statisticallysignificantly greater in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofnucleic acid expression. When nucleic acid expression is statisticallysignificantly less in the presence of the candidate compound than in itsabsence, the candidate compound is identified as an inhibitor of nucleicacid expression.

[0275] Accordingly, the invention provides methods of treatment, withthe nucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate PGP synthase nucleic acidexpression. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) oreffects on nucleic acid activity (e.g. when nucleic acid is mutated orimproperly modified). Treatment is of disorders characterized byaberrant expression or activity of the nucleic acid. Disorders that thegene is particularly relevant for treating have been disclosed hereinabove.

[0276] Alternatively, a modulator for PGP synthase nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the PGP synthase nucleic acid expression.

[0277] The PGP synthase polynucleotides are also useful for monitoringthe effectiveness of modulating compounds on the expression or activityof the PGP synthase gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

[0278] Monitoring can be, for example, as follows: (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a specified mRNA orgenomic DNA of the invention in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the niRNA or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the mRNA or genomic DNA in thepre-administration sample with the mRNA or genomic DNA in thepost-administration sample or samples; and (vi) increasing or decreasingthe administration of the agent to the subject accordingly.

[0279] The PGP synthase polynucleotides are also useful in diagnosticassays for qualitative changes in PGP synthase nucleic acid, andparticularly in qualitative changes that lead to pathology. Thepolynucleotides can be used to detect mutations in PGP synthase genesand gene expression products such as MRNA. The polynucleotides can beused as hybridization probes to detect naturally-occurring geneticmutations in the PGP synthase gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the PGP synthase geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a PGPsynthase.

[0280] Mutations in the PGP synthase gene can be detected at the nucleicacid level by a variety of techniques. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way.

[0281] In certain embodiments, detection of the mutation involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al.(1994) PNAS 91 :360-364), the latter of which can be particularly usefulfor detecting point mutations in the gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0282] It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

[0283] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well-known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0284] Alternatively, mutations in an PGP synthase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0285] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

[0286] Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature.

[0287] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method.

[0288] Furthermore, sequence differences between a mutant PGP synthasegene and a wild-type gene can be determined by direct DNA sequencing. Avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0289] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992)Meth. Enzymol. 217:286-295), electrophoretic mobility of mutant and wildtype nucleic acid is compared (Orita et al. (1989) PNAS 86:2766; Cottonet al. (1993) Mutat. Res. 285:125-144; and Hayashi et al. (1992) Genet.Anal. Tech. Appl. 9:73-79), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.(1985) Nature 313:495). The sensitivity of the assay may be enhanced byusing RNA (rather than DNA), in which the secondary structure is moresensitive to a change in sequence. In one embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5). Examples of other techniques fordetecting point mutations include, selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0290] In other embodiments, genetic mutations can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotideprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

[0291] The PGP synthase polynucleotides are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, thepolynucleotides can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). In the present case, forexample, a mutation in the PGP synthase gene that results in alteredaffinity for a coenzyme could result in an excessive or decreased drugeffect with standard concentrations of the coenzyme that activate thePGP synthase. Accordingly, the PGP synthase polynucleotides describedherein can be used to assess the mutation content of the gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment.

[0292] Thus polynucleotides displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0293] The methods can involve obtaining a control biological samplefrom a control subject, contacting the control sample with a compound oragent capable of detecting mRNA, or genomic DNA, such that the presenceof mRNA or genomic DNA is detected in the biological sample, andcomparing the presence of mRNA or genomic DNA in the control sample withthe presence of mRNA or genomic DNA in the test sample.

[0294] The PGP synthase polynucleotides are also useful for chromosomeidentification when the sequence is identified with an individualchromosome and to a particular location on the chromosome. First, theDNA sequence is matched to the chromosome by in situ or otherchromosome-specific hybridization. Sequences can also be correlated tospecific chromosomes by preparing PCR primers that can be used for PCRscreening of somatic cell hybrids containing individual chromosomes fromthe desired species. Only hybrids containing the chromosome containingthe gene homologous to the primer will yield an amplified fragment.Sublocalization can be achieved using chromosomal fragments. Otherstrategies include prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to chromosome-specific libraries. Furthermapping strategies include fluorescence in situ hybridization, whichallows hybridization with probes shorter than those traditionally used.Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on the chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0295] The PGP synthase polynucleotides can also be used to identifyindividuals from small biological samples. This can be done for exampleusing restriction fragment-length polymorphism (RFLP) to identify anindividual. Thus, the polynucleotides described herein are useful as DNAmarkers for RFLP (See U.S. Pat. No. 5,272,057).

[0296] Furthermore, the PGP synthase sequence can be used to provide analternative technique, which determines the actual DNA sequence ofselected fragments in the genome of an individual. Thus, the PGPsynthase sequences described herein can be used to prepare two PCRprimers from the 5′ and 3′ ends of the sequences. These primers can thenbe used to amplify DNA from an individual for subsequent sequencing.

[0297] Panels of corresponding DNA sequences from individuals preparedin this manner can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences. It is estimatedthat allelic variation in humans occurs with a frequency of about onceper each 500 bases. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. The PGP synthase sequences can be used to obtain suchidentification sequences from individuals and from tissue. The sequencesrepresent unique fragments of the human genome. Each of the sequencesdescribed herein can, to some degree, be used as a standard againstwhich DNA from an individual can be compared for identificationpurposes.

[0298] If a panel of reagents from the sequences is used to generate aunique identification database for an individual, those same reagentscan later be used to identify tissue from that individual. Using theunique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

[0299] The PGP synthase polynucleotides can also be used in forensicidentification procedures. PCR technology can be used to amplify DNAsequences taken from very small biological samples, such as a singlehair follicle, body fluids (e.g. blood, saliva, or semen). The amplifiedsequence can then be compared to a standard allowing identification ofthe origin of the sample.

[0300] The PGP synthase polynucleotides can thus be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As described above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to the noncoding region are particularly useful since greaterpolymorphism occurs in the noncoding regions, making it easier todifferentiate individuals using this technique.

[0301] The PGP synthase polynucleotides can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue. This is useful in cases in which a forensic pathologistis presented with a tissue of unknown origin. Panels of PGP synthaseprobes can be used to identify tissue by species and/or by organ type.

[0302] In a similar fashion, these primers and probes can be used toscreen tissue culture for contamination (i.e. screen for the presence ofa mixture of different types of cells in a culture).

[0303] Alternatively, the PGP synthase polynucleotides can be useddirectly to block transcription or translation of PGP synthase genesequences by means of antisense or ribozyme constructs. Thus, in adisorder characterized by abnormally high or undesirable PGP synthasegene expression, nucleic acids can be directly used for treatment.

[0304] The PGP synthase polynucleotides are thus useful as antisenseconstructs to control PGP synthase gene expression in cells, tissues,and organisms. A DNA antisense polynucleotide is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of PGP synthase protein.An antisense RNA or DNA polynucleotide would hybridize to the mRNA andthus block translation of mRNA into PGP synthase protein.

[0305] Examples of antisense molecules useful to inhibit nucleic acidexpression include antisense molecules complementary to a fragment ofthe 5′ untranslated region of SEQ ID NO:1, which also includes the startcodon and antisense molecules which are complementary to a fragment ofthe 3′ untranslated region of SEQ ID NO:1.

[0306] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of a PGP synthasenucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired PGP synthase nucleic acidexpression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thePGP synthase protein.

[0307] The PGP synthase polynucleotides also provide vectors for genetherapy in patients containing cells that are aberrant in PGP synthasegene expression. Thus, recombinant cells, which include the patient'scells that have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired PGPsynthase protein to treat the individual.

[0308] The invention also encompasses kits for detecting the presence ofan PGP synthase nucleic acid in a biological sample. For example, thekit can comprise reagents such as a labeled or labelable nucleic acid oragent capable of detecting PGP synthase nucleic acid in a biologicalsample; means for determining the amount of PGP synthase nucleic acid inthe sample; and means for comparing the amount of PGP synthase nucleicacid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect PGP synthase mRNA or DNA.

[0309] Computer Readable Means

[0310] The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exists in nature or in purified form.

[0311] In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

[0312] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. The skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide or amino acid sequence information of the present invention.

[0313] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

[0314] By providing the nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

[0315] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

[0316] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0317] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety ofknown algorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

[0318] For example, software which implements the BLAST (Altschul et al.(1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp.Chem. 17:203-207) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.

[0319] Vectors/Host Cells

[0320] The invention also provides vectors containing the PGP synthasepolynucleotides. The term “vector” refers to a vehicle, preferably anucleic acid molecule that can transport the PGP synthasepolynucleotides. When the vector is anucleic acid molecule, the PGPsynthase polynucleotides are covalently linked to the vector nucleicacid. With this aspect of the invention, the vector includes a plasmid,single or double stranded phage, a single or double stranded RNA or DNAviral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0321] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the PGP synthase polynucleotides. Alternatively, the vectormay integrate into the host cell genome and produce additional copies ofthe PGP synthase polynucleotides when the host cell replicates.

[0322] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the PGPsynthase polynucleotides. The vectors can function in procaryotic oreukaryotic cells or in both (shuttle vectors).

[0323] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the PGP synthase polynucleotides suchthat transcription of the polynucleotides is allowed in a host cell. Thepolynucleotides can be introduced into the host cell with a separatepolynucleotide capable of affecting transcription. Thus, the secondpolynucleotide may provide a trans-acting factor interacting with thecis-regulatory control region to allow transcription of the PGP synthasepolynucleotides from the vector. Alternatively, a transacting factor maybe supplied by the host cell. Finally, a trans-acting factor can beproduced from the vector itself.

[0324] It is understood, however, that in some embodiments,transcription and/or translation of the PGP synthase polynucleotides canoccur in a cell-free system.

[0325] The regulatory sequence to which the polynucleotides describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

[0326] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0327] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

[0328] A variety of expression vectors can be used to express a PGPsynthase polynucleotide. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0329] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0330] The PGP synthase polynucleotides can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0331] The vector containing the appropriate polynucleotide can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0332] As described herein, it may be desirable to express thepolypeptide as a fusion protein. Accordingly, the invention providesfusion vectors that allow for the production of the PGP synthasepolypeptides. Fusion vectors can increase the expression of arecombinant protein, increase the solubility of the recombinant protein,and aid in the purification of the protein by acting for example as aligand for affinity purification. A proteolytic cleavage site may beintroduced at the junction of the fusion moiety so that the desiredpolypeptide can ultimately be separated from the fusion moiety.Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al. (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) GeneExpression Technology: Methods in Enzymology 185:60-89).

[0333] Recombinant protein expression can be maximized in a hostbacteria by providing a genetic background wherein the host cell has animpaired capacity to proteolytically cleave the recombinant protein.(Gottesman, S. (1990) Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. 119-128). Alternatively, thesequence of the polynucleotide of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).

[0334] The PGP synthase polynucleotides can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J 6:229-234), pMFa (Kurjan et al. (1982) Cell 30:933-943),pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0335] The PGP synthase polynucleotides can also be expressed in insectcells using, for example, baculovirus expression vectors. Baculovirusvectors available for expression of proteins in cultured insect cells(e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. CellBiol. 3:2156-2165) and the pVL series (Lucklow et al. (1989) Virology170:31-39).

[0336] In certain embodiments of the invention, the polynucleotidesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J 6:187-195).

[0337] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the PGP synthasepolynucleotides. The person of ordinary skill in the art would be awareof other vectors suitable for maintenance propagation or expression ofthe polynucleotides described herein. These are found for example inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0338] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0339] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0340] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0341] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the PGP synthase polynucleotides can be introducedeither alone or with other polynucleotides that are not related to thePGP synthase polynucleotides such as those providing trans-actingfactors for expression vectors. When more than one vector is introducedinto a cell, the vectors can be introduced independently, co-introducedor joined to the PGP synthase polynucleotide vector.

[0342] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0343] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the polynucleotides described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0344] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0345] Where secretion of the polypeptide is desired, appropriatesecretion signals are incorporated into the vector. The signal sequencecan be endogenous to the PGP synthase polypeptides or heterologous tothese polypeptides.

[0346] Where the polypeptide is not secreted into the medium, theprotein can be isolated from the host cell by standard disruptionprocedures, including freeze thaw, sonication, mechanical disruption,use of lysing agents and the like. The polypeptide can then be recoveredand purified by well-known purification methods including ammoniumsulfate precipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

[0347] It is also understood that depending upon the host cell inrecombinant production of the polypeptides described herein, thepolypeptides can have various glycosylation patterns, depending upon thecell, or maybe non-glycosylated as when produced in bacteria. Inaddition, the polypeptides may include an initial modified methionine insome cases as a result of a host-mediated process.

[0348] Uses of Vectors and Host Cells

[0349] It is understood that “host cells” and “recombinant host cells”refer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A “purified preparation of cells”, as used herein, refers to, inthe case of plant or animal cells, an in vitro preparation of cells andnot an entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

[0350] The host cells expressing the polypeptides described herein, andparticularly recombinant host cells, have a variety of uses. First, thecells are useful for producing PGP synthase proteins or polypeptidesthat can be further purified to produce desired amounts of PGP synthaseprotein or fragments. Thus, host cells containing expression vectors areuseful for polypeptide production.

[0351] Host cells are also useful for conducting cell-based assaysinvolving the PGP synthase or PGP synthase fragments. Thus, arecombinant host cell expressing a native PGP synthase is useful toassay for compounds that stimulate or inhibit PGP synthase function.These include, but are not limited to those disclosed herein and abovein the background.

[0352] Host cells are also useful for identifying PGP synthase mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutant PGPsynthase (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native PGP synthase.

[0353] Recombinant host cells are also useful for expressing thechimeric polypeptides described herein to assess compounds that activateor suppress activation by means of a heterologous domain, segment, site,and the like, as disclosed herein.

[0354] Further, mutant PGP synthase can be designed in which one or moreof the various functions is engineered to be increased or decreased andused to augment or replace PGP synthase proteins in an individual. Thus,host cells can provide a therapeutic benefit by replacing an aberrantPGP synthase or providing an aberrant PGP synthase that provides atherapeutic result. In one embodiment, the cells provide PGP synthasethat are abnormally active.

[0355] In another embodiment, the cells provide PGP synthase that areabnormally inactive. These PGP synthase can compete with endogenous PGPsynthase in the individual.

[0356] In another embodiment, cells expressing PGP synthase that are notcatalytically active, are introduced into an individual in order tocompete with endogenous PGP synthase. For example, in the case in whichexcessive amounts of a PGP synthase substrate or effector is part of atreatment modality, it may be necessary to inactivate this molecule at aspecific point in treatment. Providing cells that compete for themolecule , but which cannot be affected by PGP synthase activation wouldbe beneficial.

[0357] Homologously recombinant host cells can also be produced thatallow the in situ alteration of endogenous PGP synthase polynucleotidesequences in a host cell genome. The host cell includes, but is notlimited to, a stable cell line, cell in vivo, or cloned microorganism.This technology is more fully described in WO 93/09222, WO 91/12650, WO91/06667, U.S. Pat. No. 5,272,071, and U.S. Pat. No. 5,641,670. Briefly,specific polynucleotide sequences corresponding to the PGP synthasepolynucleotides or sequences proximal or distal to an PGP synthase geneare allowed to integrate into a host cell genome by homologousrecombination where expression of the gene can be affected. In oneembodiment, regulatory sequences are introduced that either increase ordecrease expression of an endogenous sequence. Accordingly, a PGPsynthase protein can be produced in a cell not normally producing it.Alternatively, increased expression of PGP synthase protein can beeffected in a cell normally producing the protein at a specific level.Further, expression can be decreased or eliminated by introducing aspecific regulatory sequence. The regulatory sequence can beheterologous to the PGP synthase protein sequence or can be a homologoussequence with a desired mutation that affects expression. Alternatively,the entire gene can be deleted. The regulatory sequence can be specificto the host cell or capable of functioning in more than one cell type.Still further, specific mutations can be introduced into any desiredregion of the gene to produce mutant PGP synthase proteins. Suchmutations could be introduced, for example, into the specific functionalregions such as the ligand-binding site.

[0358] In one embodiment, the host cell can be a fertilized oocyte orembryonic stem cell that can be used to produce a transgenic animalcontaining the altered PGP synthase gene. Alternatively, the host cellcan be a stem cell or other early tissue precursor that gives rise to aspecific subset of cells and can be used to produce transgenic tissuesin an animal. See also Thomas et al., Cell 51:503 (1987) for adescription of homologous recombination vectors. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced gene has homologously recombined withthe endogenous PGP synthase gene is selected (see e.g., Li, E. et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos. WO 90/11354; WO 91/01140; and WO93/04169.

[0359] The genetically engineered host cells can be used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a PGP synthaseprotein and identifying and evaluating modulators of PGP synthaseprotein activity.

[0360] Other examples of transgenic animals include non-human primates,sheep, dogs, cows, goats, chickens, and amphibians.

[0361] In one embodiment, a host cell is a fertilized oocyte or anembryonic stem cell into which PGP synthase polynucleotide sequenceshave been introduced.

[0362] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the PGP synthase nucleotidesequences can be introduced as a transgene into the genome of anon-human animal, such as a mouse.

[0363] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the PGP synthase protein to particularcells.

[0364] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0365] In another embodiment, transgenic non-human animals can beproduced which contain selected systems, which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein is required. Such animals canbe provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase.

[0366] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0367] Transgenic animals containing recombinant cells that express thepolypeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could affect substrate bindingmay not be evident from in vitro cell-free or cell-based assays.Accordingly, it is useful to provide non-human transgenic animals toassay in vivo PGP synthase function, including substrate, cofactor andsubstituted phospho group transfer interactions. Similar methods couldbe used to determine the effect of specific mutant PGP synthase and theeffect of chimeric PGP synthase on such enzyme functions. It is alsopossible to assess the effect of null mutations, that is mutations thatsubstantially or completely eliminate one or more PGP synthasefunctions.

[0368] In general, methods for producing transgenic animals includeintroducing a nucleic acid sequence according to the present invention,the nucleic acid sequence capable of expressing the PGP synthase proteinin a transgenic animal, into a cell in culture or in vivo. Whenintroduced in vivo, the nucleic acid is introduced into an intactorganism such that one or more cell types and, accordingly, one or moretissue types, express the nucleic acid encoding the PGP synthaseprotein. Alternatively, the nucleic acid can be introduced intovirtually all cells in an organism by transfecting a cell in culture,such as an embryonic stem cell, as described herein for the productionof transgenic animals, and this cell can be used to produce an entiretransgenic organism. As described, in a further embodiment, the hostcell can be a fertilized oocyte. Such cells are then allowed to developin a female foster animal to produce the transgenic organism.

[0369] Pharmaceutical Compositions

[0370] The PGP synthase nucleic acid molecules, polypeptides andmodulators of the polypeptide and antibodies (also referred to herein as“active compounds”) can be incorporated into pharmaceutical compositionssuitable for administration to a subject, e.g., a human. Suchcompositions typically comprise the nucleic acid molecule, protein,modulator, or antibody and a pharmaceutically acceptable carrier.

[0371] The term “administer” is used in its broadest sense and includesany method of introducing the compositions of the present invention intoa subject. This includes producing polypeptides or polynucleotides invivo as by transcription or translation, in vivo, of polynucleotidesthat have been exogenously introduced into a subject. Thus, polypeptidesor nucleic acids produced in the subject from the exogenous compositionsare encompassed in the term “administer.”

[0372] As used herein the language “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions.

[0373] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdernal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

[0374] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0375] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a PGP synthase protein or anti-PGP synthaseantibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0376] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0377] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

[0378] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0379] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0380] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acidMethods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0381] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0382] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

[0383] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0384] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

[0385] The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0386] The present invention encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

[0387] It is understood that appropriate doses of small molecule agentsdepends upon a number of factors within the ken of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0388] Other Embodiments

[0389] In another aspect, the invention features, a method of analyzinga plurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a 27411preferably purified, nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to the 27411nucleic acid, polypeptide, or antibody.

[0390] The capture probes can be a set of nucleic acids from a selectedsample, e.g., a sample of nucleic acids derived from a control ornon-stimulated tissue or cell.

[0391] The method can include contacting the 27411 nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

[0392] The plurality of capture probes can be a plurality of nucleicacid probes each of which specifically hybridizes with an allele of27411. Such methods can be used to diagnose a subject, e.g., to evaluaterisk for a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder. 27411 is associated with PGP synthase activity, thus it isuseful for disorders associated with abnormal PGP synthase activity,cardiolipin biosynthesis, and PG biosynthesis.

[0393] The method can be used to detect SNPs.

[0394] In another aspect, the invention features, a method of analyzinga plurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express or mis express 27411 or from a cell orsubject in which a 27411 mediated response has been elicited, e.g., bycontact of the cell with 27411 nucleic acid or protein, oradministration to the cell or subject 27411 nucleic acid or protein;contacting the array with one or more inquiry probe, wherein an inquiryprobe can be a nucleic acid, polypeptide, or antibody (which ispreferably other than 27411 nucleic acid, polypeptide, or antibody);providing a two dimensional array having a plurality of addresses, eachaddress of the plurality being positionally distinguishable from eachother address of the plurality, and each address of the plurality havinga unique capture probe, e.g., wherein the capture probes are from a cellor subject which does not express 27411 (or does not express as highlyas in the case of the 2741 1 positive plurality of capture probes) orfrom a cell or subject which in which a 27411 mediated response has notbeen elicited (or has been elicited to a lesser extent than in the firstsample), contacting the array with one or more inquiry probes (which ispreferably other than a 27411 nucleic acid, polypeptide, or antibody),and thereby evaluating the plurality of capture probes. Binding, e.g.,in the case of a nucleic acid, hybridization with a capture probe at anaddress of the plurality, is detected, e.g., by signal generated from alabel attached to the nucleic acid, polypeptide, or antibody.

[0395] In another aspect, the invention features, a method of analyzing27411, e.g., analyzing structure, function, or relatedness to othernucleic acid or amino acid sequences. The method includes: providing a27411 nucleic acid or amino acid sequence; comparing the 27411 sequencewith one or more preferably a plurality of sequences from a collectionof sequences, e.g., a nucleic acid or protein sequence database; tothereby analyze 27411.

[0396] Preferred databases include GenBank™. The method can includeevaluating the sequence identity between a 27411 sequence and a databasesequence. The method can be performed by accessing the database at asecond site, e.g., over the internet.

[0397] In another aspect, the invention features, a set ofoligonucleotides, useful, e.g., for identifying SNP's, or identifyingspecific alleles of 27411. The set includes a plurality ofoligonucleotides, each of which has a different nucleotide at aninterrogation position, e.g., an SNP or the site of a mutation. In apreferred embodiment, the oligonucleotides of the plurality areidentical in sequence with one another (except for differences inlength). The oligonucleotides can be provided with different labels,such that an oligonucleotide that hybridizes to one allele provides asignal that is distinguishable from an oligonucleotide that hybridizesto a second allele.

[0398] This invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

EXPERIMENTAL EXAMPLE 1 Identification and Characterization of 27411,Human PGP Synthase

[0399] The human 27411 sequence (FIG. 1; SEQ ID NO:1), that isapproximately 2686 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1671nucleotides (nucleotides 315-1985 of SEQ ID NO:1; SEQ ID NO:3). Thecoding sequence encodes a 556 amino acid protein (SEQ ID NO:2).

[0400] PFAM analysis indicates that the 27411 polypeptide shares a highdegree of sequence similarity with phospholipase D domains from aminoacids 215-241 and 460-493 of SEQ ID NO:2. The phospholipase D domain(HMM) has been assigned the PFAM Accession PF00614(http://pfam.wustl.edu/). For general information regarding PFAMidentifiers, PS prefix and PF prefix domain identification numbers,refer to Sonnhammer et al. (1997) Protein 28:405-420 andhttp//www.psc.edu/general/software/packages/pfam/pfam.html.

[0401] In one embodiment, a 27411 -like protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength that spans a phospholipid membrane. More preferably, atransmembrane domain includes about at least 18, 20, 22, 24, 25, 26, or27 amino acid residues and spans a phospholipid membrane. Transmembranedomains are rich in hydrophobic residues, and typically have anα-helical structure. In a preferred embodiment, at least 50%, 60%, 70%,80%, 90%, 95% or more of the amino acids of a transmembrane domain arehydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.Transmembrane domains are described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. etal. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which areincorporated herein by reference.

[0402] In a preferred embodiment, a 27411-like polypeptide or proteinhas at least one transmembrane domain or a region which includes atleast 18, 20, 22, 24, 25, 26, or 27 amino acid residues and has at leastabout 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with a“transmembrane domain,” e.g., at least one transmembrane domain of human27411-like (e.g., amino acid residues 51 to 73 or 469 to 485 of SEQ IDNO:2).

[0403] In another embodiment, a 2741 1-like protein includes at leastone “non-transmembrane domain.” As used herein, “non-transmembranedomains” are domains that reside outside of the membrane. When referringto plasma membranes, non-transmembrane domains include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes and microsomes), non-transmembrane domains include thosedomains of the protein that reside in the cytosol (i.e., the cytoplasm),the lumen of the organelle, or the matrix or the intermembrane space(the latter two relate specifically to mitochondria organelles). TheC-terminal amino acid residue of a non-transmembrane domain is adjacentto an N-terminal amino acid residue of a transmembrane domain in anaturally occurring 27411-like, or 27411 -like protein.

[0404] In a preferred embodiment, a 27411 -like polypeptide or proteinhas a “non-transmembrane domain” or a region which includes at leastabout 1-396, preferably about 100-396, more preferably about 200-350,and even more preferably about 240-280 amino acid residues, and has atleast about 60%, 70% 80% 90% 95%, 99% or 100% sequence identity with a“non-transmembrane domain”, e.g., a non-transmembrane domain of human27411-like (e.g., residues 1 to 51, 74 to 468, and 486 to 556 of SEQ IDNO:2). Preferably, a non-transmembrane domain is capable of catalyticactivity (e.g., PGP synthase).

[0405] A non-transmembrane domain located at the N-terminus of a 27411-like protein or polypeptide is referred to herein as an “N-terminalnon-transmembrane domain.” As used herein, an “N-terminalnon-transmembrane domain” includes an amino acid sequence having about1-51, preferably about 10-45, more preferably about 20-40, or even morepreferably about 20-35 amino acid residues in length and is locatedoutside the boundaries of a membrane. For example, an N-terminalnon-transmembrane domain is located at about amino acid residues 1 to 50of SEQ ID NO:2.

[0406] Similarly, a non-transmembrane domain located at the C-terminusof 27411 -like protein or polypeptide is referred to herein as a“C-terminal non-transmembrane domain.” As used herein, an “C-terminalnon-transmembrane domain” includes an amino acid sequence having about1-7 1, preferably about 10-75, preferably about 20-60, more preferablyabout 25-45 amino acid residues in length and is located outside theboundaries of a membrane. For example, an C-terminal non-transmembranedomain is located at about amino acid residues 486 to 556 of SEQ IDNO:2.

[0407] A 27411-like molecule can further include a signal sequence. Asused herein, a “signal sequence” refers to a peptide of about 20-80amino acid residues in length which occurs at the N-terminus ofsecretory and integral membrane proteins and which contains a majorityof hydrophobic amino acid residues. For example, a signal sequencecontains at least about 12-25 amino acid residues, preferably about30-70 amino acid residues, more preferably about 68 amino acid residues,and has at least about 40-70%, preferably about 50-65%, and morepreferably about 55-60% hydrophobic amino acid residues (e.g., alanine,valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, orproline). Such a “signal sequence”, also referred to in the art as a“signal peptide”, serves to direct a protein containing such a sequenceto a lipid bilayer. For example, in one embodiment, a 27411-like proteincontains a signal sequence of about amino acids 1 to 68 of SEQ ID NO:2.The “signal sequence” may be cleaved during processing of the matureprotein. The mature 2741 1-like protein corresponds to amino acids 69 to556 of SEQ ID NO:2.

[0408] The 27411 protein displays approximately 26% identity from aa85-522 to a ProDom consensus sequence found in O-phosphatidyltransferaseCDP-diacylglycerol serine phosphatidylserine synthase transferasephospholipid biosynthesis; approximately 31% identity from aa 476-554 toa ProDom consensus sequence found in receptor nuclear co-repressor N-corretinoid X interacting protein; approximately 31% identity from aa260-324 to a ProDom consensus sequence found in SIP1 proteinphosphorylation; and, approximately 38% identity from aa 210-247 to aProDom consensus sequence found in protein transferase HP019transmembrane CSGC-MDOG intergenic region. These sequences wereidentified by the ProDom program, which is available from INRA, GREG(107/94), MESR (ACC-SV13), the CNRS “Genome Initiative” and the EuropeanUnion. The ProDom Program (http://www.toulouse.inra.fr/prodom.html)allows analysis of domain arrangements in proteins and protein families.A detailed description of ProDom analysis can be found in Corpet et al.(1999) Nuc. Acids Res. 27:263-267.

EXAMPLE 2 Tissue Distribution of 27411 mRNA

[0409] Expression levels of 27411 in various tissue and cell types weredetermined by quantitative RT-PCR (Reverse Transcriptase PolymeraseChain Reaction; Taqman® brand PCR kit, Applied Biosystems). Thequantitative RT-PCR reactions were performed according to the kitmanufacturer's instructions. The results of the Taqman® analysis areshown in FIG. 5.

[0410] TaqMan analysis of 27411 revealed expression in a number oftissues, including the following: artery, diseased artery, vein,coronary smooth muscle cells, HUVEC (umbilical vein endothelial cells),hemangioma, heart, congestive heart failure heart, kidney, skeletalmuscle, adipose, pancreas, primary osteoblasts, differentiatedosteoclasts, skin, spinal cord, brain cortex, brain hypothalamus, nerve,dorsal root ganglion, breast, breast tumor, ovary, ovarian tumor,prostate, prostate tumor, salivary glands, colon, colon tumor, lung,lung tumor, chronic obstructive pulmonary disease lung, inflammatorybowel disease colon, liver, liver fibrosis, spleen, tonsil, lymph node,small intestine, macrophages, synovium, mononuclear bone marrow cells,activated peripheral blood mononuclear cells, neutrophils,megakaryocytes, and erythroid tissue.

[0411] Northern blot hybridizations with various RNA samples areperformed under standard conditions and washed under stringentconditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all ora portion of the 27411 cDNA (SEQ ID NO:1) can be used. The DNA isradioactively labeled with ³²P-dCTP using the Prime-It Kit (Stratagene,La Jolla, Calif.) according to the instructions of the supplier. Filterscontaining mRNA from mouse hematopoietic and endocrine tissues, andcancer cell lines (Clontech, Palo Alto, Calif.) are probed in ExpressHybhybridization solution (Clontech) and washed at high stringencyaccording to manufacturer's recommendations.

EXAMPLE 3 Recombinant Expression of 27411 in Bacterial Cells

[0412] In this example, 27411 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 27411 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-27411 fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

EXAMPLE 4 Expression of Recombinant 27411 Protein in COS Cells

[0413] To express the 27411 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 27411 protein and an HA tag (Wilson et al (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

[0414] To construct the plasmid, the 27411 DNA sequence is amplified byPCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 27411coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 27411 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 27411 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

[0415] COS cells are subsequently transfected with the 27411-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the 27411 polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

[0416] Alternatively, DNA containing the 27411 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 27411polypeptide is detected by radiolabelling and immunoprecipitation usinga 27411 specific monoclonal antibody.

[0417] This invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will fully conveythe invention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 3 <210> SEQ ID NO 1 <211>LENGTH: 2686 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (315)...(1985) <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(2686) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 1 atccacgctt ttgcntgacc cttgcttggt tcaacttanaggtctttgtt tcggttttct 60 tgttnngcnc cggttacaga tccaaagttt tgaaaaaaccanaaaagtna nctggtaagt 120 taagtctttt ttgtctttta tttccagntc cnggaatccgggtggttggt gcaaantcaa 180 aaganttgtt cctcaagtga atgttgcntt tacttcttaggcntgtacgg aaagtgttat 240 ttttgtttta aaagctggga attcttanta cgacttcactatagggagtc gacccacgcg 300 tccggcgagt ctcc atg gcg gtg gcg gcg gca gctgcg gcg gga ccc gtg 350 Met Ala Val Ala Ala Ala Ala Ala Ala Gly Pro Val1 5 10 ttc tgg agg cga ctg ctg ggc ctc ctg cct ggc cgc cca ggg ctg gcc398 Phe Trp Arg Arg Leu Leu Gly Leu Leu Pro Gly Arg Pro Gly Leu Ala 1520 25 gcg ctc ctg gga cgc ctg tcc gac cgc ctc ggc agg aac cgg gac cgc446 Ala Leu Leu Gly Arg Leu Ser Asp Arg Leu Gly Arg Asn Arg Asp Arg 3035 40 cag cgc agg agg tca cca tgg ctg tta ttg gct ccc ttg ctg tcc cca494 Gln Arg Arg Arg Ser Pro Trp Leu Leu Leu Ala Pro Leu Leu Ser Pro 4550 55 60 gct gtt ccc cag gtc acc tcc cca cct tgc tgc ctg tgt cca gaa ggc542 Ala Val Pro Gln Val Thr Ser Pro Pro Cys Cys Leu Cys Pro Glu Gly 6570 75 gtg cac cgg ttc cag tgg atc aga aac ctg gtt cca gaa ttt gga gtc590 Val His Arg Phe Gln Trp Ile Arg Asn Leu Val Pro Glu Phe Gly Val 8085 90 tcc agt tct cac gtt agg gtg ctt tct tcc ccg gca gag ttt ttc gag638 Ser Ser Ser His Val Arg Val Leu Ser Ser Pro Ala Glu Phe Phe Glu 95100 105 ctc atg aag ggg cag ata aga gta gcc aag agg cgg gtc gtg atg gca686 Leu Met Lys Gly Gln Ile Arg Val Ala Lys Arg Arg Val Val Met Ala 110115 120 tcc ctc tac ctg ggg aca ggt cct ttg gaa cag gag ctg gtg gac tgc734 Ser Leu Tyr Leu Gly Thr Gly Pro Leu Glu Gln Glu Leu Val Asp Cys 125130 135 140 ctg gaa agt act cta gaa aag tca ctc caa gca aag ttt cct tcaaat 782 Leu Glu Ser Thr Leu Glu Lys Ser Leu Gln Ala Lys Phe Pro Ser Asn145 150 155 ctc aag gtc tcc att ctc tta gac ttc acg cgg ggc tca cga ggtcgg 830 Leu Lys Val Ser Ile Leu Leu Asp Phe Thr Arg Gly Ser Arg Gly Arg160 165 170 aag aac tcc cgc aca atg ctg ctc cca ctc ctg cgg agg ttc ccagag 878 Lys Asn Ser Arg Thr Met Leu Leu Pro Leu Leu Arg Arg Phe Pro Glu175 180 185 cag gtc cga gtc tcc ctc ttt cac acg ccg cac ctc cgt ggg ctgctt 926 Gln Val Arg Val Ser Leu Phe His Thr Pro His Leu Arg Gly Leu Leu190 195 200 cgg ctc ctc atc cct gag cgc ttc aac gag acc atc ggc ctc cagcac 974 Arg Leu Leu Ile Pro Glu Arg Phe Asn Glu Thr Ile Gly Leu Gln His205 210 215 220 att aag gtg tac ctc ttc gac aac agc gtc atc ttg agc ggtgca aac 1022 Ile Lys Val Tyr Leu Phe Asp Asn Ser Val Ile Leu Ser Gly AlaAsn 225 230 235 ctg agt gac tcc tac ttc acc aac cgc cag gac cgc tac gtgttc ctg 1070 Leu Ser Asp Ser Tyr Phe Thr Asn Arg Gln Asp Arg Tyr Val PheLeu 240 245 250 cag gac tgt gcg gag att gcc gac ttc ttc acg gag ctg gtggac gcg 1118 Gln Asp Cys Ala Glu Ile Ala Asp Phe Phe Thr Glu Leu Val AspAla 255 260 265 gtg ggg gat gtg tcc ctg cag ctg cag ggg gac gac acg gtgcag gtg 1166 Val Gly Asp Val Ser Leu Gln Leu Gln Gly Asp Asp Thr Val GlnVal 270 275 280 gtg gat ggg atg gtg cat cct tac aaa ggg gac cgg gcc gagtac tgc 1214 Val Asp Gly Met Val His Pro Tyr Lys Gly Asp Arg Ala Glu TyrCys 285 290 295 300 aag gca gcc aat aag agg gtc atg gat gtg atc aac tcagcc agg acc 1262 Lys Ala Ala Asn Lys Arg Val Met Asp Val Ile Asn Ser AlaArg Thr 305 310 315 cgc cag cag atg ctg cat gcc cag acc ttc cac agc aactct ctt ttg 1310 Arg Gln Gln Met Leu His Ala Gln Thr Phe His Ser Asn SerLeu Leu 320 325 330 acc cag gaa gat gca gca gct gct ggg gat cgc aga ccagcc cct gac 1358 Thr Gln Glu Asp Ala Ala Ala Ala Gly Asp Arg Arg Pro AlaPro Asp 335 340 345 acc tgg att tat ccg ctg att cag atg aag ccc ttc gagatt caa atc 1406 Thr Trp Ile Tyr Pro Leu Ile Gln Met Lys Pro Phe Glu IleGln Ile 350 355 360 gat gag att gtc act gag acc ctg ttg act gag gcg gagcgc ggg gca 1454 Asp Glu Ile Val Thr Glu Thr Leu Leu Thr Glu Ala Glu ArgGly Ala 365 370 375 380 aag gtc tac ctc acc act ggc tat ttc aac ctg acccag gcc tac atg 1502 Lys Val Tyr Leu Thr Thr Gly Tyr Phe Asn Leu Thr GlnAla Tyr Met 385 390 395 gac ctg gtc ttg ggc act cgg gct gag tac cag atcctg ctg gcc tca 1550 Asp Leu Val Leu Gly Thr Arg Ala Glu Tyr Gln Ile LeuLeu Ala Ser 400 405 410 cca gag gtg aat ggc ttc ttt ggg gcc aag ggg gtggcc ggc gcc atc 1598 Pro Glu Val Asn Gly Phe Phe Gly Ala Lys Gly Val AlaGly Ala Ile 415 420 425 cca gcg gcc tat gtg cac atc gag cga cag ttc ttcagt gag gtg tgc 1646 Pro Ala Ala Tyr Val His Ile Glu Arg Gln Phe Phe SerGlu Val Cys 430 435 440 agc ctg gga cag cag gag cgg gtc cag ctt cag gagtac tgg cgg agg 1694 Ser Leu Gly Gln Gln Glu Arg Val Gln Leu Gln Glu TyrTrp Arg Arg 445 450 455 460 ggc tgg acg ttc cac gcc aaa ggc ctc tgg ctgtac ctg gca ggg agc 1742 Gly Trp Thr Phe His Ala Lys Gly Leu Trp Leu TyrLeu Ala Gly Ser 465 470 475 agc ctg ccc tgt ctc acg ctg att ggc tct cctaat ttt ggg tac agg 1790 Ser Leu Pro Cys Leu Thr Leu Ile Gly Ser Pro AsnPhe Gly Tyr Arg 480 485 490 tca gtt cac cgg gac ctg gag gcc cag att gcgatc gtg acg gag aac 1838 Ser Val His Arg Asp Leu Glu Ala Gln Ile Ala IleVal Thr Glu Asn 495 500 505 cag gcc ctg cag cag cag ctt cac cag gag caagag cag ctc tac ctg 1886 Gln Ala Leu Gln Gln Gln Leu His Gln Glu Gln GluGln Leu Tyr Leu 510 515 520 agg tca ggt gtg gtg tcc tct gcc acc ttc gagcag ccg agt cgc cag 1934 Arg Ser Gly Val Val Ser Ser Ala Thr Phe Glu GlnPro Ser Arg Gln 525 530 535 540 gtg aag ctg tgg gtg aag atg gtg act ccactg atc aag aac ttc ttc 1982 Val Lys Leu Trp Val Lys Met Val Thr Pro LeuIle Lys Asn Phe Phe 545 550 555 tga ggacagacag gtgctgtctc tagcatcacctctcagcacg attttcccga 2035 * gagttcacag gaatggcctt gatgaagatg acaggcatggccggggtcag ctctttcagc 2095 cgcgcttcag cgatgactcc agtctgggtg tcccagcgagcccctgcagg gacagtatgg 2155 ctgagggtca ggtgtgctgc cagtaagtga gggaggggctggcaggaagg gtggggtcct 2215 cacactcccc gccctytgca gagctgggct ctaccccaaaaggcttcagg ccagctgcca 2275 cagctggaag cagaggcctt cgtaggtgat ggcctgcatgttgtaactac cccgtcccgc 2335 tgggctcaag gaacagctca gctaaagccc tcgggttccatccgtttaaa tctgtggcat 2395 tttcagagcc tcatctgtca gccttaatgt cagtggcaggaagtcataac tccagctaaa 2455 aattacagag taaagttccc tgattcttaa tgtgtaatgtctgccctatg tgtacataca 2515 caatataatt atacatctgt gcatataaat attgcctttaaccagactgc tattatttct 2575 actcgcccta tttaatggtg tttttatttc ctgtctgaaatctcaaaata aacaaacatg 2635 gagagcttaa aaaaaaaaaa aaagggcggc cgctagactagtctagagaa a 2686 <210> SEQ ID NO 2 <211> LENGTH: 556 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ala Val Ala Ala AlaAla Ala Ala Gly Pro Val Phe Trp Arg Arg 1 5 10 15 Leu Leu Gly Leu LeuPro Gly Arg Pro Gly Leu Ala Ala Leu Leu Gly 20 25 30 Arg Leu Ser Asp ArgLeu Gly Arg Asn Arg Asp Arg Gln Arg Arg Arg 35 40 45 Ser Pro Trp Leu LeuLeu Ala Pro Leu Leu Ser Pro Ala Val Pro Gln 50 55 60 Val Thr Ser Pro ProCys Cys Leu Cys Pro Glu Gly Val His Arg Phe 65 70 75 80 Gln Trp Ile ArgAsn Leu Val Pro Glu Phe Gly Val Ser Ser Ser His 85 90 95 Val Arg Val LeuSer Ser Pro Ala Glu Phe Phe Glu Leu Met Lys Gly 100 105 110 Gln Ile ArgVal Ala Lys Arg Arg Val Val Met Ala Ser Leu Tyr Leu 115 120 125 Gly ThrGly Pro Leu Glu Gln Glu Leu Val Asp Cys Leu Glu Ser Thr 130 135 140 LeuGlu Lys Ser Leu Gln Ala Lys Phe Pro Ser Asn Leu Lys Val Ser 145 150 155160 Ile Leu Leu Asp Phe Thr Arg Gly Ser Arg Gly Arg Lys Asn Ser Arg 165170 175 Thr Met Leu Leu Pro Leu Leu Arg Arg Phe Pro Glu Gln Val Arg Val180 185 190 Ser Leu Phe His Thr Pro His Leu Arg Gly Leu Leu Arg Leu LeuIle 195 200 205 Pro Glu Arg Phe Asn Glu Thr Ile Gly Leu Gln His Ile LysVal Tyr 210 215 220 Leu Phe Asp Asn Ser Val Ile Leu Ser Gly Ala Asn LeuSer Asp Ser 225 230 235 240 Tyr Phe Thr Asn Arg Gln Asp Arg Tyr Val PheLeu Gln Asp Cys Ala 245 250 255 Glu Ile Ala Asp Phe Phe Thr Glu Leu ValAsp Ala Val Gly Asp Val 260 265 270 Ser Leu Gln Leu Gln Gly Asp Asp ThrVal Gln Val Val Asp Gly Met 275 280 285 Val His Pro Tyr Lys Gly Asp ArgAla Glu Tyr Cys Lys Ala Ala Asn 290 295 300 Lys Arg Val Met Asp Val IleAsn Ser Ala Arg Thr Arg Gln Gln Met 305 310 315 320 Leu His Ala Gln ThrPhe His Ser Asn Ser Leu Leu Thr Gln Glu Asp 325 330 335 Ala Ala Ala AlaGly Asp Arg Arg Pro Ala Pro Asp Thr Trp Ile Tyr 340 345 350 Pro Leu IleGln Met Lys Pro Phe Glu Ile Gln Ile Asp Glu Ile Val 355 360 365 Thr GluThr Leu Leu Thr Glu Ala Glu Arg Gly Ala Lys Val Tyr Leu 370 375 380 ThrThr Gly Tyr Phe Asn Leu Thr Gln Ala Tyr Met Asp Leu Val Leu 385 390 395400 Gly Thr Arg Ala Glu Tyr Gln Ile Leu Leu Ala Ser Pro Glu Val Asn 405410 415 Gly Phe Phe Gly Ala Lys Gly Val Ala Gly Ala Ile Pro Ala Ala Tyr420 425 430 Val His Ile Glu Arg Gln Phe Phe Ser Glu Val Cys Ser Leu GlyGln 435 440 445 Gln Glu Arg Val Gln Leu Gln Glu Tyr Trp Arg Arg Gly TrpThr Phe 450 455 460 His Ala Lys Gly Leu Trp Leu Tyr Leu Ala Gly Ser SerLeu Pro Cys 465 470 475 480 Leu Thr Leu Ile Gly Ser Pro Asn Phe Gly TyrArg Ser Val His Arg 485 490 495 Asp Leu Glu Ala Gln Ile Ala Ile Val ThrGlu Asn Gln Ala Leu Gln 500 505 510 Gln Gln Leu His Gln Glu Gln Glu GlnLeu Tyr Leu Arg Ser Gly Val 515 520 525 Val Ser Ser Ala Thr Phe Glu GlnPro Ser Arg Gln Val Lys Leu Trp 530 535 540 Val Lys Met Val Thr Pro LeuIle Lys Asn Phe Phe 545 550 555 <210> SEQ ID NO 3 <211> LENGTH: 1671<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(1671) <400> SEQUENCE: 3 atg gcg gtggcg gcg gca gct gcg gcg gga ccc gtg ttc tgg agg cga 48 Met Ala Val AlaAla Ala Ala Ala Ala Gly Pro Val Phe Trp Arg Arg 1 5 10 15 ctg ctg ggcctc ctg cct ggc cgc cca ggg ctg gcc gcg ctc ctg gga 96 Leu Leu Gly LeuLeu Pro Gly Arg Pro Gly Leu Ala Ala Leu Leu Gly 20 25 30 cgc ctg tcc gaccgc ctc ggc agg aac cgg gac cgc cag cgc agg agg 144 Arg Leu Ser Asp ArgLeu Gly Arg Asn Arg Asp Arg Gln Arg Arg Arg 35 40 45 tca cca tgg ctg ttattg gct ccc ttg ctg tcc cca gct gtt ccc cag 192 Ser Pro Trp Leu Leu LeuAla Pro Leu Leu Ser Pro Ala Val Pro Gln 50 55 60 gtc acc tcc cca cct tgctgc ctg tgt cca gaa ggc gtg cac cgg ttc 240 Val Thr Ser Pro Pro Cys CysLeu Cys Pro Glu Gly Val His Arg Phe 65 70 75 80 cag tgg atc aga aac ctggtt cca gaa ttt gga gtc tcc agt tct cac 288 Gln Trp Ile Arg Asn Leu ValPro Glu Phe Gly Val Ser Ser Ser His 85 90 95 gtt agg gtg ctt tct tcc ccggca gag ttt ttc gag ctc atg aag ggg 336 Val Arg Val Leu Ser Ser Pro AlaGlu Phe Phe Glu Leu Met Lys Gly 100 105 110 cag ata aga gta gcc aag aggcgg gtc gtg atg gca tcc ctc tac ctg 384 Gln Ile Arg Val Ala Lys Arg ArgVal Val Met Ala Ser Leu Tyr Leu 115 120 125 ggg aca ggt cct ttg gaa caggag ctg gtg gac tgc ctg gaa agt act 432 Gly Thr Gly Pro Leu Glu Gln GluLeu Val Asp Cys Leu Glu Ser Thr 130 135 140 cta gaa aag tca ctc caa gcaaag ttt cct tca aat ctc aag gtc tcc 480 Leu Glu Lys Ser Leu Gln Ala LysPhe Pro Ser Asn Leu Lys Val Ser 145 150 155 160 att ctc tta gac ttc acgcgg ggc tca cga ggt cgg aag aac tcc cgc 528 Ile Leu Leu Asp Phe Thr ArgGly Ser Arg Gly Arg Lys Asn Ser Arg 165 170 175 aca atg ctg ctc cca ctcctg cgg agg ttc cca gag cag gtc cga gtc 576 Thr Met Leu Leu Pro Leu LeuArg Arg Phe Pro Glu Gln Val Arg Val 180 185 190 tcc ctc ttt cac acg ccgcac ctc cgt ggg ctg ctt cgg ctc ctc atc 624 Ser Leu Phe His Thr Pro HisLeu Arg Gly Leu Leu Arg Leu Leu Ile 195 200 205 cct gag cgc ttc aac gagacc atc ggc ctc cag cac att aag gtg tac 672 Pro Glu Arg Phe Asn Glu ThrIle Gly Leu Gln His Ile Lys Val Tyr 210 215 220 ctc ttc gac aac agc gtcatc ttg agc ggt gca aac ctg agt gac tcc 720 Leu Phe Asp Asn Ser Val IleLeu Ser Gly Ala Asn Leu Ser Asp Ser 225 230 235 240 tac ttc acc aac cgccag gac cgc tac gtg ttc ctg cag gac tgt gcg 768 Tyr Phe Thr Asn Arg GlnAsp Arg Tyr Val Phe Leu Gln Asp Cys Ala 245 250 255 gag att gcc gac ttcttc acg gag ctg gtg gac gcg gtg ggg gat gtg 816 Glu Ile Ala Asp Phe PheThr Glu Leu Val Asp Ala Val Gly Asp Val 260 265 270 tcc ctg cag ctg cagggg gac gac acg gtg cag gtg gtg gat ggg atg 864 Ser Leu Gln Leu Gln GlyAsp Asp Thr Val Gln Val Val Asp Gly Met 275 280 285 gtg cat cct tac aaaggg gac cgg gcc gag tac tgc aag gca gcc aat 912 Val His Pro Tyr Lys GlyAsp Arg Ala Glu Tyr Cys Lys Ala Ala Asn 290 295 300 aag agg gtc atg gatgtg atc aac tca gcc agg acc cgc cag cag atg 960 Lys Arg Val Met Asp ValIle Asn Ser Ala Arg Thr Arg Gln Gln Met 305 310 315 320 ctg cat gcc cagacc ttc cac agc aac tct ctt ttg acc cag gaa gat 1008 Leu His Ala Gln ThrPhe His Ser Asn Ser Leu Leu Thr Gln Glu Asp 325 330 335 gca gca gct gctggg gat cgc aga cca gcc cct gac acc tgg att tat 1056 Ala Ala Ala Ala GlyAsp Arg Arg Pro Ala Pro Asp Thr Trp Ile Tyr 340 345 350 ccg ctg att cagatg aag ccc ttc gag att caa atc gat gag att gtc 1104 Pro Leu Ile Gln MetLys Pro Phe Glu Ile Gln Ile Asp Glu Ile Val 355 360 365 act gag acc ctgttg act gag gcg gag cgc ggg gca aag gtc tac ctc 1152 Thr Glu Thr Leu LeuThr Glu Ala Glu Arg Gly Ala Lys Val Tyr Leu 370 375 380 acc act ggc tatttc aac ctg acc cag gcc tac atg gac ctg gtc ttg 1200 Thr Thr Gly Tyr PheAsn Leu Thr Gln Ala Tyr Met Asp Leu Val Leu 385 390 395 400 ggc act cgggct gag tac cag atc ctg ctg gcc tca cca gag gtg aat 1248 Gly Thr Arg AlaGlu Tyr Gln Ile Leu Leu Ala Ser Pro Glu Val Asn 405 410 415 ggc ttc tttggg gcc aag ggg gtg gcc ggc gcc atc cca gcg gcc tat 1296 Gly Phe Phe GlyAla Lys Gly Val Ala Gly Ala Ile Pro Ala Ala Tyr 420 425 430 gtg cac atcgag cga cag ttc ttc agt gag gtg tgc agc ctg gga cag 1344 Val His Ile GluArg Gln Phe Phe Ser Glu Val Cys Ser Leu Gly Gln 435 440 445 cag gag cgggtc cag ctt cag gag tac tgg cgg agg ggc tgg acg ttc 1392 Gln Glu Arg ValGln Leu Gln Glu Tyr Trp Arg Arg Gly Trp Thr Phe 450 455 460 cac gcc aaaggc ctc tgg ctg tac ctg gca ggg agc agc ctg ccc tgt 1440 His Ala Lys GlyLeu Trp Leu Tyr Leu Ala Gly Ser Ser Leu Pro Cys 465 470 475 480 ctc acgctg att ggc tct cct aat ttt ggg tac agg tca gtt cac cgg 1488 Leu Thr LeuIle Gly Ser Pro Asn Phe Gly Tyr Arg Ser Val His Arg 485 490 495 gac ctggag gcc cag att gcg atc gtg acg gag aac cag gcc ctg cag 1536 Asp Leu GluAla Gln Ile Ala Ile Val Thr Glu Asn Gln Ala Leu Gln 500 505 510 cag cagctt cac cag gag caa gag cag ctc tac ctg agg tca ggt gtg 1584 Gln Gln LeuHis Gln Glu Gln Glu Gln Leu Tyr Leu Arg Ser Gly Val 515 520 525 gtg tcctct gcc acc ttc gag cag ccg agt cgc cag gtg aag ctg tgg 1632 Val Ser SerAla Thr Phe Glu Gln Pro Ser Arg Gln Val Lys Leu Trp 530 535 540 gtg aagatg gtg act cca ctg atc aag aac ttc ttc tga 1671 Val Lys Met Val Thr ProLeu Ile Lys Asn Phe Phe * 545 550 555

That which is claimed:
 1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequences of the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein said nucleotide sequence encodes a polypeptide having biological activity; b) a nucleic acid molecule comprising a fragment of at least 20 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequences of the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-20 11 or PTA-2340, e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a biologically active polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising the complement of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions; and f) a nucleic acid molecule comprising the complement of a), b), c), d), or e).
 2. The isolated nucleic acid molecule of claim 1 , which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the nucleotide sequences of the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, or a complement thereof.
 3. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.
 4. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide.
 5. A host cell which contains the nucleic acid molecule of claim 1 .
 6. The host cell of claim 5 which is a mammalian host cell.
 7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1 .
 8. An isolated polypeptide selected from the group consisting of: a) a biologically active polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequences of the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising the complement of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions; and, c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2; and d) a polypeptide having at least 60% sequence identity to the amino acid sequence SEQ ID NO:2, wherein the polypeptide has biological activity.
 9. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO:2.
 10. The polypeptide of claim 8 further comprising heterologous amino acid sequences.
 11. An antibody which selectively binds to a polypeptide of claim 8 .
 12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-201 1 or PTA-2340, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-20 1 or PTA-2340; c) a biologically active naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequences encoded by the cDNA inserts of the plasmids deposited with ATCC as Patent Deposit Numbers PTA-2011 or PTA-2340, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising the complement of SEQ ID NO:1 or SEQ ID NO:3, d) a polypeptide having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide has biological activity; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.
 13. A method for detecting the presence of a polypeptide of claim 8 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8 ; and b) determining whether the compound binds to the polypeptide in the sample.
 14. The method of claim 13 , wherein the compound which binds to the polypeptide is an antibody.
 15. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use.
 16. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
 17. The method of claim 16 , wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
 18. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
 19. A method for identifying a compound which binds to a polypeptide of claim 8 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 8 with a test compound; and b) determining whether the polypeptide binds to the test compound.
 20. The method of claim 19 , wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for PGP synthase-mediated phospho-group transfer.
 21. A method for modulating the activity of a polypeptide of claim 8 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 8 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
 22. A method for identifying a compound which modulates the activity of a polypeptide of claim 8 , comprising: a) contacting a polypeptide of claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound that modulates the activity of the polypeptide. 